Peptides and compounds that bind to a receptor

ABSTRACT

Described are peptides and peptide mimetics that bind to and activate the thrombopoietin receptor. Such peptides and peptide mimetics are useful in methods for treating hematological disorders and particularly, thrombocytopenia resulting from chemotherapy, radiation therapy, or bone marrow transfusions as well as in diagnostic methods employing labeled peptides and peptide mimetics.

CROSS REFERENCE TO RELATED CASES

This application is a continuation-in-part of Ser. No. 08/699,027, filedAug. 15, 1996, now abandoned, which is a CIP of InternationalApplication No. PCT/US96/09623, filed Jun. 7, 1996, Ser. No. 08/485,301,filed Jun. 7, 1995, now abandoned and Ser. No. 08/478,128, filed Jun. 7,1995, now abandoned.

BACKGROUND OF THE INVENTION

The present invention provides peptides and compounds that bind to andactivate the thrombopoietin receptor (c-mpl or TPO-R) or otherwise actas a TPO agonist. The invention has application in the fields ofbiochemistry and medicinal chemistry and particularly provides TPOagonists for use in the treatment of human disease.

Megakaryocytes are bone marrow-derived cells, which are responsible forproducing circulating blood platelets. Although comprising <0.25% of thebone marrow cells in most species, they have >10 times the volume oftypical marrow cells. See Kuter, et. al., Proc. Natl. Acad. Sci. USA91:11104-11108 (1994). Megakaryocytes undergo a process known asendomitosis whereby they replicate their nuclei but fail to undergo celldivision and thereby give rise to polyploid cells. In response to adecreased platelet count, the endomitotic rate increases, higher ploidymegakaryocytes are formed, and the number of megakaryocytes may increaseup to 3-fold. See Harker, J. Clin. Invest., 47:458-465 (1968). Incontrast, in response to an elevated platelet count, the endomitoticrate decreases, lower ploidy megakaryocytes are formed, and the numberof megakaryocytes may decrease by 50%.

The exact physiological feedback mechanism by which the mass ofcirculating platelets regulates the endomitotic rate and number of bonemarrow megakaryocytes is not known. The circulating thrombopoieticfactor involved in mediating this feedback loop is now thought to bethrombopoietin (TPO). More specifically, TPO has been shown to be themain humoral regulator in situations involving thrombocytopenia. See,e.g., Metcalf, Nature, 369:519-520 (1994). TPO has been shown in severalstudies to increase platelet counts, increase platelet size, andincrease isotope incorporation into platelets of recipient animals.Specifically, TPO is thought to affect megakaryocytopoiesis in severalways: (1) it produces increases in megakaryocyte size and number; (2) itproduces an increase in DNA content, in the form of polyploidy, inmegakaryocytes; (3) it increases megakaryocyte endomitosis; (4) itproduces increased maturation of megakaryocytes; and (5) it produces anincrease in the percentage of precursor cells, in the form of smallacetylcholinesterase-positive cells, in the bone marrow.

Because platelets (thrombocytes) are necessary for blood clotting andwhen their numbers are very low a patient is at serious risk of deathfrom catastrophic hemorrhage, TPO has potential useful application inboth the diagnosis and the treatment of various hematological disorders,for example, diseases primarily due to platelet defects. Ongoingclinical trials with TPO have indicated that TPO can be administeredsafely to patients. In addition, recent studies have provided a basisfor the projection of efficacy of TPO therapy in the treatment ofthrombocytopenia, and particularly thrombocytopenia resulting fromchemotherapy, radiation therapy, or bone marrow transplantation astreatment for cancer or lymphoma. See, e.g., McDonald, Am. J. Ped.Hematology/Oncology, 14:8-21 (1992).

The gene encoding TPO has been cloned and characterized. See Kuter, etal., Proc. Natl. Acad. Sci. USA, 91:11104-11108 (1994); Barley, et al.,Cell 77:1117-1124 (1994); Kaushansky et al., Nature 369:568-571 (1994);Wendling, et al., Nature, 369:571-574 (1994); and Sauvage et al., Nature369:533-538 (1994). Thrombopoietin is a glycoprotein with at least twoforms, with apparent molecular masses of 25 kDa and 31 kDa, with acommon N-terminal amino acid sequence. See, Bartley, et al., Cell,77:1117-1124 (1994). Thrombopoietin appears to have two distinct regionsseparated by a potential Arg-Arg cleavage site. The amino-terminalregion is highly conserved in man and mouse, and has some homology witherythropoietin and interferon-a and interferon-b. The carboxy-terminalregion shows wide species divergence.

The DNA sequences and encoded peptide sequences for human TPO-R (alsoknown as c-mpl) have been described. See Vigon, et al., Proc. Natl.Acad. Sci. USA, 89:5640-5644 (1992). TPO-R is a member of thehematopoietin growth factor receptor family, a family characterized by acommon structural design of the extracellular domain, including fourconserved C residues in the N-terminal portion and a WSXWS motif (SEQ IDNO:1) close to the transmembrane region. See Bazan, Proc. Natl. Acad.Sci. USA, 87:6934-6938 (1990). Evidence that this receptor plays afunctional role in hematopoiesis includes observations that itsexpression is restricted to spleen, bone marrow, or fetal liver in mice(see Souyri, et al., Cell 63:1137-1147 (1990)) and to megakaryocytes,platelets, and CD34⁺ cells in humans (see Methia, et al., Blood82:1395-1401 (1993)). Furthermore, exposure of CD34⁺ cells to syntheticoligonucleotides antisense to mpl RNA significantly inhibits theappearance of megakaryocyte colonies without affecting erythroid ormyeloid colony formation. Some workers postulate that the receptorfunctions as a homodimer, similar to the situation with the receptorsfor G-CSF and erythropoietin.

The availability of cloned genes for TPO-R facilitates the search foragonists of this important receptor. The availability of the recombinantreceptor protein allows the study of receptor-ligand interaction in avariety of random and semi-random peptide diversity generation systems.These systems include the "peptides on plasmids" system described inU.S. Pat. Nos. 5,270,170 and 5,338,665; the "peptides on phage" systemdescribed in U.S. patent application Ser. No. 07/718,577, filed Jun. 20,1991, U.S. patent application Ser. No. 07/541,108, filed Jun. 20, 1990,and in Cwirla, et al., Proc. Natl. Acad. Sci. USA, 87:6378-6382 (1990);the "polysome" system described in U.S. patent application Ser. No.08/300,262, filed Sep. 2, 1994, which is a continuation-in-partapplication based on U.S. patent application Ser. No. 08/144,775, filedOct. 29, 1993 and PCT WO 95/11992; the "encoded synthetic library"system described in U.S. patent application Ser. No. 08/146,886, filedNov. 12, 1993, Ser. No. 07/946,239, filed Sep. 16, 1992, and Ser. No.07/762,522, filed Sep. 18, 1991; and the "very large scale immobilizedpolymer synthesis" system described in U.S. Pat. No. 5,143,854; PCTPatent Publication No. 90/15070, published Dec. 13, 1990; U.S. patentapplication Ser. No. 07/624,120, filed Dec. 6, 1990; Fodor, et al.,Science, 251:767-773 (2/1991); Dower, et al., Ann. Rep. Med. Chem.,26:271-180 (1991); and U.S. patent application Ser. No. 07/805,727,filed Dec. 6, 1991; each of the foregoing patent applications andpublications is incorporated herein by reference.

The slow recovery of platelet levels in patients suffering fromthrombocytopenia is a serious problem, and has lent urgency to thesearch for a blood growth factor agonist able to accelerate plateletregeneration. The present invention provides such an agonist.

SUMMARY OF THE INVENTION

This invention is directed, in part, to the novel and unexpecteddiscovery that defined low molecular weight peptides and peptidemimetics have strong binding properties to the TPO-R and can activatethe TPO-R. Accordingly, such peptides and peptide mimetics are usefulfor therapeutic purposes in treating conditions mediated by TPO (e.g.,thrombocytopenia resulting from chemotherapy, radiation therapy, or bonemarrow transfusions) as well as for diagnostic purposes in studying themechanism of hematopoiesis and for the in vitro expansion ofmegakaroycytes and committed progenitor cells.

Peptides and peptide mimetics suitable for therapeutic and/or diagnosticpurposes have an IC₅₀ of about 2 mM or less, as determined by thebinding affinity assay set forth in Example 3 below wherein a lower IC₅₀correlates to a stronger binding affinity to TPO-R. For pharmaceuticalpurposes, the peptides and peptidomimetics preferably have an IC₅₀ of nomore than about 100 μm, more preferably, no more than 500 nM. In apreferred embodiment, the molecular weight of the peptide or peptidemimetic is from about 250 to about 8,000 daltons. If the peptides ofthis invention are oligomerized, dimerized and/or derivatized with ahydrophilic polymer as described herein, the molecular weights of suchpeptides will be substantially greater and can range anywhere from about500 to about 120,000 daltons, more preferable from about 8,000 to about80,000 daltons.

When used for diagnostic purposes, the peptides and peptide mimeticspreferably are labeled with a detectable label and, accordingly, thepeptides and peptide mimetics without such a label serve asintermediates in the preparation of labeled peptides and peptidemimetics.

Peptides meeting the defined criteria for molecular weight and bindingaffinity for TPO-R comprise 9 or more amino acids wherein the aminoacids are naturally occurring or synthetic (non-naturally occurring)amino acids. Peptide mimetics include peptides having one or more of thefollowing modifications:

peptides wherein one or more of the peptidyl --C(O)NR--! linkages(bonds) have been replaced by a non-peptidyl linkage such as a --CH₂-carbamate linkage --CH₂ --OC(O)NR--!; a phosphonate linkage; a --CH₂-sulfonamide --CH₂ --S(O)₂ NR--! linkage; a urea --NHC(O)NH--! linkage;a --CH₂ -secondary amine linkage; or an alkylated peptidyl linkage--C(O)NR⁶ -- where R⁶ is lower alkyl!;

peptides wherein the N-terminus is derivatized to a --NRR¹ group; to a--NRC(O)R group; to a --NRC(O)OR group; to a --NRS(O)₂ R group; to a--NHC(O)NHR group where R and R¹ are hydrogen or lower alkyl with theproviso that R and R¹ are not both hydrogen; to a succinimide group; toa benzyloxycarbonyl-NH-- (CBZ--NH--) group; or to abenzyloxycarbonyl-NH-- group having from 1 to 3 substituents on thephenyl ring selected from the group consisting of lower alkyl, loweralkoxy, chloro, and bromo; or peptides wherein the C terminus isderivatized to --C(O)R² where ² is selected from the group consisting oflower alkoxy, and --NR³ R⁴ where R³ and R⁴ are independently selectedfrom the group consisting of hydrogen and lower alkyl.

Accordingly, preferred peptides and peptide mimetics comprise a compoundhaving:

(1) a molecular weight of less than about 5000 daltons, and

(2) a binding affinity to TPO-R as expressed by an IC₅₀ of no more thanabout 100 μm,

wherein from zero to all of the --C(O)NH-- linkages of the peptide havebeen replaced by a linkage selected from the group consisting of a --CH₂OC(O)NR-- linkage; a phosphonate linkage; a --CH₂ S(O)₂ NR-- linkage; a--CH₂ NR-- linkage; and a --C(O)NR⁶ -- linkage; and a --NHC(O)NH--linkage where R is hydrogen or lower alkyl and R⁶ is lower alkyl,

further wherein the N-terminus of said peptide or peptide mimetic isselected from the group consisting of a --NRR¹ group; a --NRC(O)R group;a --NRC(O)OR group; a --NRS(O)₂ R group; a --NHC(O)NHR group; asuccinimide group; a benzyloxycarbonyl-NH-- group; and abenzyloxycarbonyl-NH-- group having from 1 to 3 substituents on thephenyl ring selected from the group consisting of lower alkyl, loweralkoxy, chloro, and bromo, where R and R¹ are independently selectedfrom the group consisting of hydrogen and lower alkyl,

and still further wherein the C-terminus of said peptide or peptidemimetic has the formula --C(O)R² where R² is selected from the groupconsisting of hydroxy, lower alkoxy, and --NR³ R⁴ where R³ and R⁴ areindependently selected from the group consisting of hydrogen and loweralkyl and where the nitrogen atom of the --NR³ R⁴ group can optionallybe the amine group of the N-terminus of the peptide so as to form acyclic peptide,

and physiologically acceptable salts thereof.

In a related embodiment, the invention is directed to a labeled peptideor peptide mimetic comprising a peptide or peptide mimetic described asabove having covalently attached thereto a label capable of detection.

In some embodiments of the invention, preferred peptides for use includepeptides having a core structure comprising a sequence of amino acids:(SEQ ID NO:2)

    X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6 X.sub.7

where X₁ is C, L, M, P, Q, V; X₂ is F, K, L, N, Q, R, S, T or V; X₃ isC, F, I, L, M, R, S, V or W; X₄ is any of the 20 genetically codedL-amino acids; X₅ is A, D, E, G, K, M, Q, R, S, T, V or Y; X₆ is C, F,G, L, M, S, V, W or Y; and X₇ is C, G, I, K, L, M, N, R or V.

In a preferred embodiment the core peptide comprises a sequence of aminoacids: (SEQ ID NO: 3)

    X.sub.8 G X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 W X.sub.7

where X₁ is L, M, P, Q, or V; X₂ is F, R, S, or T; X₃ is F, L, V, or W;X₄ is A, K, L, M, R, S, V, or T; X₅ is A, E, G, K, M, Q, R, S, or T; X₇is C, I, K, L, M or V; and each X₈ residue is independently selectedfrom any of the 20 genetically coded L-amino acids, their stereoisomericD-amino acids; and non-natural amino acids. Preferably, each X₈ residueis independently selected from any of the 20 genetically coded L-aminoacids and their stereoisomeric D-amino acids. In a preferred embodiment(SEQ ID NO:4), X₁ is P; X₂ is T; X₃ is L; X₄ is R; X₅ is E or Q; and X₇is I or L.

More preferably, the core peptide comprises a sequence of amino acids:(SEQ ID NO:5)

    X.sub.9 X.sub.8 G X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 W X.sub.7

where X₉ is A, C, E, G, I, L , M, P, R, Q, S, T, or V; and X₈ is A, C,D, E, K, L, Q, R, S, T, or V. More preferably, X₉ is A or I; and X₈ isD, E, or K.

Particularly preferred peptides include: (SEQ ID NOS 6-13, respectively)G G C A D G P T L R E W I S F C G G; G N A D G P T L R Q W L E G R R P KN; G G C A D G P T L R E W I S F C G G K; T I K G P T L R Q W L K S R EH T S; S I E G P T L R E W L T S R T P H S; L A I E G P T L R Q W L H GN G R D T; C A D G P T L R E W I S F C; and I E G P T L R Q W L A A R A.

In further embodiments of the invention, preferred peptides for use inthis invention include peptides having a core structure comprising asequence of amino acids: (SEQ ID NO: 14)

    C X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6 X.sub.7

where X₂ is F, K, L, N, Q, R, S, T or V; X₃ is C, F, I, L, M, R, S or V;X₄ is any of the 20 genetically coded L-amino acids; X₅ is A, D, E, G,S, V or Y; X₆ is C, F, G, L, M, S, V, W or Y; and X₇ is C, G, I, K, L,M, N, R or V. In a more preferred embodiment, X₄ is A, E, G, H, K, L, M,P, Q, R, S, T, or W. In a further embodiment, X₂ is S or T; X₃ is L orR; X₄ is R; X₅ is D, E, or G; X₆ is F, L, or W; and X₇ is I, K, L, R, orV. Particularly preferred peptides include: (SEQ ID NO: 15) G G C T L RE W L H G G F C G G.

In a further embodiment, preferred peptides for use in this inventioninclude peptides having a structure comprising a sequence of aminoacids: (SEQ ID NO: 16)

    X.sub.8 C X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6 X.sub.7

where X₂ is F, K, L, N, Q, R, S, T or V; X₃ is C, F, I, L, M, R, S, V orW; X₄ is any of the 20 genetically coded L-amino acids; X₅ is A, D, E,G, K, M, Q, R, S, T, V or Y; X₆ is C, F, G, L, M, S, V, W or Y; X₇ is C,G, I, K, L, M, N, R or V; and X₈ is any of the 20 genetically codedL-amino acids. In some embodiments, X₈ is preferably G, S, Y, or R.

In another embodiment, the peptide compounds of the present inventionare preferably dimerized or oligomerized to increase the affinity and/oractivity of the compounds. Examples of preferred dimerized peptidecompounds include, but are not limited to, the following: ##STR1##

In yet a further embodiment, preferred peptides for use in thisinvention include peptides that are covalently attached to one or moreof a variety of hydrophilic polymers. Suitable hydrophilic polymersinclude, but are not limited to, polyalkylethers as exemplified bypolyethylene glycol and polypropylene glycol, polylactic acid,polyglycolic acid, polyoxyalkenes, polyvinylalcohol,polyvinylpyrrolidone, cellulose and cellulose derivatives, dextran anddextran derivatives, etc. It has surprisingly been discovered that whenthe peptide compounds are derivatized with such polymers, theirsolubility and circulation half-lives are increased with little, if any,diminishment in their binding activity. In a presently preferredembodiment, the peptide compounds of this invention are dimerized andeach of the dimeric subunits is covalently attached to a hydrophilicpolymer. In a further preferred embodiment, the peptide compounds ofthis invention are PEGylated, i.e., covalently attached to polyethyleneglycol (PEG). Examples of preferred PEGylated, dimerized peptidecompositions of this invention include, but are not limited to, thefollowing: ##STR2## wherein "n" is an interger having a value rangingfrom about 5 to about 1000, more preferably from about 10 to about 600and, even more preferably, from about 110 to about 450.

The compounds described herein are useful for the prevention andtreatment of diseases mediated by TPO, and particularly for treatinghematological disorders, including but not limited to, thrombocytopeniaresulting from chemotherapy, radiation therapy, or bone marrowtransfusions. Thus, the present invention also provides a method fortreating wherein a patient having a disorder that is susceptible totreatment with a TPO agonist receives, or is administered, atherapeutically effective dose or amount of a compound of the presentinvention.

The invention also provides for pharmaceutical compositions comprisingone or more of the compounds described herein and a physiologicallyacceptable carrier. These pharmaceutical compositions can be in avariety of forms including oral dosage forms, as well as inhalablepowders and solutions and injectable and infusible solutions.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-B illustrates the results of a functional assay in the presenceof various peptides; the assay is described in Example 2. FIG. 1A is agraphical depiction of the results of the TPO-R transfected Ba/F3 cellproliferation assay for selected peptides of the invention:

▪ designating the results for (SEQ ID NO: 8) G G C A D G P T L R E W I SF C G G K (biotin);

X designating the results for (SEQ ID NO:6) G G C A D G P T L R E W I SF C G G;

▴ designating the results for (SEQ ID NO: 11) L A I E G P T L R Q W L HG N G R D T;

◯ designating the results for (SEQ ID NO:7) G N A D G P T L R Q W L E GR R P K N; and

+ designating the results for (SEQ ID NO:9) T I K G P T L R Q W L K S RE H T S.

FIG. 1B is a graphical depiction of the results with the same peptidesand the parental cell line.

FIGS. 2A-C show the results of peptide oligomerization using the TPO-Rtransfected Ba/F3 cell proliferation assay. FIG. 2A shows the results ofthe assay for the complexed biotinylated peptide (AF 12285 withstreptavidin (SA)) for both the transfected and parental cell lines.FIG. 2B shows the results of the assay for the free biotinylated peptide(AF 12285) for both the transfected and parental cell lines. FIG. 2Cshows the results of the assay for streptavidin alone for both thetransfected and parental cell lines.

FIGS. 3A-G show the results of a series of control experiments showingthe activity of TPO, the peptides of the present invention, EPO, andEPO-R binding peptides in a cell proliferation assay using either theTPO-R transfected Ba/F3 cell line and its corresponding parental line,or an EPO-dependent cell line. FIG. 3A depicts the results for TPO inthe cell proliferation assay using the TPO-R transfected Ba/F3 cell lineand its corresponding parental line. FIG. 3B depicts the results for EPOin the cell proliferation assay using the TPO-R transfected Ba/F3 cellline and its corresponding parental line. FIG. 3C depicts the resultsfor complexed biotinylated peptide (AF 12285 with streptavidin (SA)) anda complexed form of a biotinylated EPO-R binding peptide (AF 11505 withSA) in the TPO-R transfected Ba/F3 cell line. The results for thecorresponding parental cell line are shown in FIG. 3D. FIG. 3E depictsthe results for TPO in the cell proliferation assay using theEPO-dependent cell line. FIG. 3F depicts the results for EPO in the cellproliferation assay using the EPO-dependent cell line. FIG. 3G depictsthe results for complexed biotinylated peptide (AF 12885 withstreptavidin (SA)) and the completed form of a biotinylated EPO-Rbinding peptide (AF 11505 with SA) in the EPO-dependent cell line.

FIGS. 4A-C illustrates the construction of peptides-on-plasmidslibraries in vector pJS142. FIG. 4A shows a restriction map and positionof the genes. The library plasmid includes the rrnB transcriptionalterminator, the bla gene to permit selection on ampicillin, the M13phage intragenic region (M13 IG) to permit rescue of single-strandedDNA, a plasmid replication origin (ori), two lacO_(s) sequences, and thearaC gene to permit positive and negative regulation of the araBpromoter driving expression of the lac fusion gene. FIG. 4B (SEQ ID NOS233-234, respectively) shows the sequence of the cloning region at the3' end of the lac I gene, including the SfiI and EagI sites used duringlibrary construction. FIG. 4C (SEQ ID NOS 235-236, respectively) showsthe ligation of annealed library oligonucleotides, ON-829 and ON-830, toSfiI sites of pJS142 to produce a library. Single spaces in the sequenceindicate sites of ligation.

FIGS. 5A-B illustrate cloning into the pELM3 and pELM15 MBP vectors.FIG. 5A (SEQ ID NOS 237-238, respectively) shows the sequence at the 3'end of the ma1E fusion gene, including the MBP coding sequence, the polyasparagine linker, the factor Xa protease cleavage site, and theavailable cloning sites. The remaining portions of the vectors arederived from pMALc2 (pELM3) and pMALp2 (pELM15), available from NewEngland Biolabs. FIG. 5B (SEQ ID NOS 239-240, respectively) shows thesequence of the vectors after transfer of the BspEII-ScaI libraryfragment into AgeI-ScaI digested pELM3/pELM15. The transferred sequenceincludes the sequence encoding the GGG peptide linker from the pJS142library.

FIG. 6A depicts a restriction map and position of the genes for theconstruction of headpiece dimer libraries in vector pCMG14. The libraryplasmid includes: the rrnb transcriptional terminator, the bla gene topermit selection on ampicillin, the M13 phage intragenic region (M13 IG)to permit rescue of single-stranded DNA, a plasmid replication origin(ori), one lacO_(s) sequence, and the araC gene to permit positive andnegative regulation of the araB promoter driving expression of theheadpiece diner fusion gene. FIG. 6B (SEQ ID NOS 241-242, respectively)depicts the sequence of the cloning region at the 3' end of theheadpiece dimer gene, including the SfiI and EagI sites used duringlibrary construction. FIG. 6C (SEQ ID NOS: 234-244, respectively) showsthe ligation of annealed ON-1679, ON-829, and ON-830 to SfiI sites ofpCMG14 to produce a library. Singles spaces in the sequence indicatesites of ligation.

FIGS. 7-9 show the results of further assays evaluating activity of thepeptides and peptide mimetics of the invention. In this assay mice aremade thrombocytopenic with carboplatin. FIG. 7 depicts typical resultswhen Balb/C mice are treated with carboplatin (125 mg/kgintraperitoneally) on Day 0. The dashed lines represent untreatedanimals from three experiments. The solid line representcarboplatin-treated groups in three experiments. The heavy solid linesrepresent historical data. FIG. 8 depicts the effect of carboplatintitration on platelet counts in mice treated with the indicated amountsof carboplatin (in mg/kg, intraperitoneally (ip) on Day 0). FIG. 9depicts amelioration of carboplatin-induced thrombocytopenia on Day 10by peptide AF12513 (513). Carboplatin (CBP; 50-125 mg/kg,intraperitoneally) was administered on Day 0. AF12513 (1 mg/kg, ip) wasgiven on Days 1-9.

FIG. 10 illustrates the plasma concentration of 5K PEG-AF13948, 20KPEG-AF13948 and vehicle AF13948, 5K PEG-AF13948 and vehicle following IVadministration of 700 μg/kg in mice.

FIG. 11 illustrates the plasma concentration of 5K PEG-AF13948, 20KPEG-AF13948 and vehicle following IV administration of 500 μg/kg inmice.

FIG. 12 illustrates the plasma concentrations of 20K DiPEG-AF13948following IV injection of 100, 10, and 1 μg/kg in mice.

FIG. 13 illustrates the plasma concentration of AF13948-20K dePEGfollowing IV and SC administration of 10 μg/kg to mice. As can be seefrom FIGS. 10-13, PEGylation of the peptide compound results in theirimproved pharmacokinetic characteristics.

FIG. 14 illustrates the stability of AF13948 and GW350781 in human serumand demonstrates that the PEGylated compound has increased stability,i.e., increased half-life, over the non-PEGylated compound.

FIGS. 15-17 (SEQ ID NOS 193 & 18, respectively) illustrate exemplarreactions schemes for derivatizing the peptide compounds of the presentinvention with, for example, polyethylene glycol (PEG).

FIG. 18 illustrates the pharmacokinetic profiles of a peptide compoundof the present invention variously derivatized with PEG. In thisexperiment, the peptide AF13948 was derivatized with branched PEG(di(2)), with an ester linked PEG (SPA) and with an aldehyde linked PEG(ALDH) (see, FIGS. 15-17). The results obtained indicate that all threeof the peptide compounds variously derivatized with PEG have favorablepharmacokinetic profiles.

FIGS. 19-20 illustrate the effects of the PEGylated peptide compounds ofthe present invention on carboplatin (CBP)-induced thrombocytopenia inmice. FIG. 19 demonstrates that GW350781, the 5K-PEGylated peptide, canameliorate thrombocytopenia in a mouse model. FIG. 20 demonstrates thatGW305805, the 20K-diPEGylated peptide, can also amelioratecarboplatin-induced thrombocytopenia and that it is even more potentthan the 5K-PEGylated peptide.

FIGS. 21-22 illustrate the effects of GW350781 and GW350805 onthrombocytosis in normal mice. The results obtained indicate that thePEGylated peptide compounds of the invention have a favorable effectthrombocytosis, with the 20K-diPEGylated peptide being about 100-foldmore potent than the 5K-PEGylated peptide.

FIGS. 23-24 illustrate the effects of varying doses of GW350781 andGW350805 on platelet levels in normal mice. Such data demonstrate thatthe PEGylated peptide compounds of the invention can increase plateletlevels in normal mice.

FIG. 25 illustrates the effect of single-dose vs. multiple-dose ofGW305805 on platelet levels in normal mice.

FIG. 26 illustrates a general synthetic scheme for making dimer peptideswith β-Ala.

FIG. 27 illustrates a general synthetic scheme for making diner peptideswithout β-Ala.

DESCRIPTION OF SPECIFIC EMBODIMENTS

I. DEFINITIONS AND GENERAL PARAMETERS

The following definitions are set forth to illustrate and define themeaning and scope of the various terms used to describe the inventionherein.

"Agonist" refers to a biologically active ligand which binds to itscomplementary biologically active receptor and activates the lattereither to cause a biological response in the receptor or to enhancepreexisting biological activity of the receptor.

"Pharmaceutically acceptable salts" refer to the non-toxic alkali metal,alkaline earth metal, and ammonium salts commonly used in thepharmaceutical industry including the sodium, potassium, lithium,calcium, magnesium, barium, ammonium, and protamine zinc salts, whichare prepared by methods well known in the art. The term also includesnon-toxic acid addition salts, which are generally prepared by reactingthe compounds of this invention with a suitable organic or inorganicacid. Representative salts include the hydrochloride, hydrobromide,sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, borate,benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,succinate, tartrate, napsylate, and the like.

"Pharmaceutically acceptable acid addition salt" refers to those saltswhich retain the biological effectiveness and properties of the freebases and which are not biologically or otherwise undesirable, formedwith inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid and the like, and organicacids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, malic acid, malonic acid, succinic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, menthanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like. For a descriptionof pharmaceutically acceptable acid addition salts as prodrugs, seeBundgaard, H., supra.

"Pharmaceutically acceptable ester" refers to those esters which retain,upon hydrolysis of the ester bond, the biological effectiveness andproperties of the carboxylic acid or alcohol and are not biologically orotherwise undesirable. For a description of pharmaceutically acceptableesters as prodrugs, see Bundgaard, H., ed., Design of Prodrugs, ElsevierScience Publishers, Amsterdam (1985). These esters are typically formedfrom the corresponding carboxylic acid and an alcohol. Generally, esterformation can be accomplished via conventional synthetic techniques.(See, e.g., March, Advanced Organic Chemistry, 4th Ed., John Wiley &Sons, New York (1992), 393-396 and references cited therein, and Mark,et al., Encyclopedia of Chemical Technology, John Wiley & Sons, New York(1980).) The alcohol component of the ester will generally comprise (i)a C₂ -C₁₂ aliphatic alcohol that can or can not contain one or moredouble bonds and can or can not contain branched carbons or (ii) a C₇-C₁₂ aromatic or heteroaromatic alcohols. This invention alsocontemplates the use of those compositions which are both esters asdescribed herein and at the same time are the pharmaceuticallyacceptable acid addition salts thereof.

"Pharmaceutically acceptable amide" refers to those amides which retain,upon hydrolysis of the amide bond, the biological effectiveness andproperties of the carboxylic acid or amine and are not biologically orotherwise undesirable. For a description of pharmaceutically acceptableamides as prodrugs, see Bundgaard, H., ed., Design of Prodrugs, ElsevierScience Publishers, Amsterdam (1985). These amides are typically formedfrom the corresponding carboxylic acid and an amine. Generally, amideformation can be accomplished via conventional synthetic techniques.(See, e.g., March, Advanced Organic Chemistry, 4th Ed., John Wiley &Sons, New York (1992), p. 393 and Mark, et al. Encyclopedia of ChemicalTechnology, John Wiley & Sons, New York (1980).) This invention alsocontemplates the use of those compositions which are both amides asdescribed herein and at the same time are the pharmaceuticallyacceptable acid addition salts thereof.

"Pharmaceutically or therapeutically acceptable carrier" refers to acarrier medium which does not interfere with the effectiveness of thebiological activity of the active ingredients and which is not toxic tothe host or patient.

"Stereoisomer" refers to a chemical compound having the same molecularweight, chemical composition, and constitution as another, but with theatoms grouped differently. That is, certain identical chemical moietiesare at different orientations in space and, therefore, when pure, hasthe ability to rotate the plane of polarized light. However, some purestereoisomers may have an optical rotation that is so slight that it isundetectable with present instrumentation. The compounds of the instantinvention may have one or more asymmetrical carbon atoms and thereforeinclude various stereoisomers. All stereoisomers are included within thescope of the invention.

"Therapeutically- or pharmaceutically-effective amount" as applied tothe compositions of the instant invention refers to the amount ofcomposition sufficient to induce a desired biological result. Thatresult can be alleviation of the signs, symptoms, or causes of adisease, or any other desired alteration of a biological system. In thepresent invention, the result will typically involve a decrease in theimmunological and/or inflammatory responses to infection or tissueinjury.

Amino acid residues in peptides are abbreviated as follows:Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I;Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Prolineis Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyror Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn orN; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Gluor E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg orR; and Glycine is Gly or G. Additionally, t-Buo is tert-bulyloxy, Bzl isbenzyl, CHA is cyclohexylamine, Ac is acetyl, Me is methyl, Pen ispenicillamine, Aib is aminoisobutyric acid, Nva is norvaline, Abu isaminobutyric acid, Thi is thienylalanine, OBn is O-benzyl, and hyp ishydroxyproline.

In addition to peptides consisting only of naturally-occurring aminoacids, peptidomimetics or peptide analogs are also provided. Peptideanalogs are commonly used in the pharmaceutical industry as non-peptidedrugs with properties analogous to those of the template peptide. Thesetypes of non-peptide compound are termed "peptide mimetics" or"peptidomimetics" (Luthman, et al., A Textbook of Drug Design andDevelopment, 14:386-406, 2nd Ed., Harwood Academic Publishers (1996);Joachim Grante, Angew. Chem. Int. Ed. Engl., 33:1699-1720 (1994);Fauchere, J., Adv. Drug Res., 15:29 (1986); Veber and Freidinger TINS,p. 392 (1985); and Evans, et al., J. Med. Chem. 30:1229 (1987), whichare incorporated herein by reference). Peptide mimetics that arestructurally similar to therapeutically useful peptides may be used toproduce an equivalent or enhanced therapeutic or prophylactic effect.Generally, peptidomimetics are structurally similar to a paradigmpolypeptide (i.e., a polypeptide that has a biological orpharmacological activity), such as naturally-occurring receptor-bindingpolypeptide, but have one or more peptide linkages optionally replacedby a linkage selected from the group consisting of: --CH₂ NH--, --CH₂S--, --CH₂ --CH₂ --, --CH═CH-- (cis and trans), --COCH₂ --, --CH(OH)CH₂--, and --CH₂ SO--, by methods known in the art and further described inthe following references: Spatola, A. F. in Chemistry and Biochemistryof Amino Acids, Peptides, and Proteins, B. Weinstein, eds., MarcelDekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983),Vol. 1, Issue 3, Peptide Backbone Modifications (general review);Morley, Trends Pharm. Sci. pp. 463-468 (1980), (general review); Hudson,et al., Int. J. Pept. Prot. Res., 14:177-185 (1979) (--CH₂ NH--, CH₂ CH₂--); Spatola, et al., Life Sci., 38:1243-1249 (1986) (--CH₂ --S); Hann,J. Chem. Soc. Perkin Trans. I, 307-314 (1982) (--CH--CH--, cis andtrans); Almquist, et al., J. Med. Chem., 23:1392-1398, (1980) (--COCH₂--); Jennings-White, et al., Tetrahedron Lett. 23:2533 (1982) (--COCH₂--); Szelke, et al., European Appln. EP 45665 (1982) (--CH(OH)CH₂ --);Holladay, et al., Tetrahedron Lett., 24:4401-4404 (1983) (--C(OH)CH₂--); and Hruby, Life Sci., 31:189-199 (1982) (--CH₂ --S--); each ofwhich is incorporated herein by reference. A particularly preferrednon-peptide linkage is --CH₂ NH--. Such peptide mimetics may havesignificant advantages over polypeptide embodiments, including, forexample: more economical production, greater chemical stability,enhanced pharmacological properties (half-life, absorption, potency,efficacy, etc.), altered specificity (e.g., a broad-spectrum ofbiological activities), reduced antigenicity, and others. Labeling ofpeptidomimetics usually involves covalent attachment of one or morelabels, directly or through a spacer (e.g., an amide group), tonon-interfering position(s) on the peptidomimetic that are predicted byquantitative structure-activity data and/or molecular modeling. Suchnon-interfering positions generally are positions that do not formdirect contacts with the macromolecules(s) (e.g., immunoglobulinsuperfamily molecules) to which the peptidomimetic binds to produce thetherapeutic effect. Derivitization (e.g., labeling) of peptidomimeticsshould not substantially interfere with the desired biological orpharmacological activity of the peptidomimetic. Generally,peptidomimetics of receptor-binding peptides bind to the receptor withhigh affinity and possess detectable biological activity (i.e., areagonistic or antagonistic to one or more receptor-mediated phenotypicchanges).

Systematic substitution of one or more amino acids of a consensussequence with a D-amino acid of the same type (e.g., D-lysine in placeof L-lysine) may be used to generate more stable peptides. In addition,constrained peptides comprising a consensus sequence or a substantiallyidentical consensus sequence variation may be generated by methods knownin the art (Rizo, et al., Ann. Rev. Biochem., 61:387 (1992),incorporated herein by reference); for example, by adding internalcysteine residues capable of forming intramolecular disulfide bridgeswhich cyclize the peptide.

Synthetic or non-naturally occurring amino acids refer to amino acidswhich do not naturally occur in vivo but which, nevertheless, can beincorporated into the peptide structures described herein. Preferredsynthetic amino acids are the D-α-amino acids of naturally occurringL-α-amino acid as well as non-naturally occurring D- and L-α-amino acidsrepresented by the formula H₂ NCHR⁵ COOH where R⁵ is 1) a lower alkylgroup, 2) a cycloalkyl group of from 3 to 7 carbon atoms, 3) aheterocycle of from 3 to 7 carbon atoms and 1 to 2 heteroatoms selectedfrom the group consisting of oxygen, sulfur, and nitrogen, 4) anaromatic residue of from 6 to 10 carbon atoms optionally having from 1to 3 substituents on the aromatic nucleus selected from the groupconsisting of hydroxyl, lower alkoxy, amino, and carboxyl, 5)-alkylene-Y where alkylene is an alkylene group of from 1 to 7 carbonatoms and Y is selected from the group consisting of (a) hydroxy, (b)amino, (c) cycloalkyl and cycloalkenyl of from 3 to 7 carbon atoms, (d)aryl of from 6 to 10 carbon atoms optionally having from 1 to 3substituents on the aromatic nucleus selected from the group consistingof hydroxyl, lower alkoxy, amino and carboxyl, (e) heterocyclic of from3 to 7 carbon atoms and 1 to 2 heteroatoms selected from the groupconsisting of oxygen, sulfur, and nitrogen, (f) --C(O)R² where R² isselected from the group consisting of hydrogen, hydroxy, lower alkyl,lower alkoxy, and --NR³ R⁴ where R³ and R⁴ are independently selectedfrom the group consisting of hydrogen and lower alkyl, (g) --S(O)_(n) R⁶where n is an integer from 1 to 2 and R⁶ is lower alkyl and with theproviso that R⁵ does not define a side chain of a naturally occurringamino acid.

Other preferred synthetic amino acids include amino acids wherein theamino group is separated from the carboxyl group by more than one carbonatom such as β-alanine, γ-aminobutyric acid, and the like.

Particularly preferred synthetic amino acids include, by way of example,the D-amino acids of naturally occurring L-amino acids,L-(1-naphthyl)-alanine, L-(2-naphthyl)-alanine, L-cyclohexylalanine,L-2-aminoisobutyric acid, the sulfoxide and sulfone derivatives ofmethionine (i.e., HOOC--(H₂ NCH)CH₂ CH₂ --S(O)_(n) R⁶) where n and R₆are as defined above as well as the lower alkoxy derivative ofmethionine (i.e., HOOC--(H₂ NCH)CH₂ CH₂ --OR⁶ where R⁶ is as definedabove).

"Detectable label" refers to materials, which when covalently attachedto the peptides and peptide mimetics of this invention, permit detectionof the peptide and peptide mimetics in vivo in the patient to whom thepeptide or peptide mimetic has been administered. Suitable detectablelabels are well known in the art and include, by way of example,radioisotopes, fluorescent labels (e.g., fluorescein), and the like. Theparticular detectable label employed is not critical and is selectedrelative to the amount of label to be employed as well as the toxicityof the label at the amount of label employed. Selection of the labelrelative to such factors is well within the skill of the art.

Covalent attachment of the detectable label to the peptide or peptidemimetic is accomplished by conventional methods well known in the art.For example, when the ¹²⁵ I radioisotope is employed as the detectablelabel, covalent attachment of ¹²⁵ I to the peptide or the peptidemimetic can be achieved by incorporating the amino acid tyrosine intothe peptide or peptide mimetic and then iodimating the peptide (see,e.g., Weaner, et al., Synthesis and Applications of IsotopicallyLabelled Compounds, pp. 137-140 (1994)). If tyrosine is not present inthe peptide or peptide mimetic, incorporation of tyrosine to the N or Cterminus of the peptide or peptide mimetic can be achieved by well knownchemistry. Likewise, ³² P can be incorporated onto the peptide orpeptide mimetic as a phosphate moiety through, for example, a hydroxylgroup on the peptide or peptide mimetic using conventional chemistry.

II. OVERVIEW

The present invention provides compounds that bind to and activate theTPO-R or otherwise behave as a TPO agonist. These compounds include"lead" peptide compounds and "derivative" compounds constructed so as tohave the same or similar molecular structure or shape as the leadcompounds but that differ from the lead compounds either with respect tosusceptibility to hydrolysis or proteolysis and/or with respect to otherbiological properties, such as increased affinity for the receptor. Thepresent invention also provides compositions comprising an effectiveamount of a TPO agonist, and more particularly a compound, that isuseful for treating hematological disorders, and particularly,thrombocytopenia associated with chemotherapy, radiation therapy, orbone marrow transfusions.

III. IDENTIFICATION OF TPO-AGONISTS

Peptides having a binding affinity to TPO-R can be readily identified byrandom peptide diversity generating systems coupled with an affinityenrichment process.

Specifically, random peptide diversity generating systems include the"peptides on plasmids" system described in U.S. Pat. Nos. 5,270,170 and5,338,665; the "peptides on phage" system described in U.S. patentapplication Ser. No. 07/718,577, filed Jun. 20, 1991 which is acontinuation in part application of U.S. patent application Ser. No.07/541,108, filed Jun. 20, 1990, and in Cwirla, et al., Proc. Natl.Acad. Sci. USA, 87:6378-6382 (1990); the "polysome system" described inU.S. patent application Ser. No. 08/300,262, filed Sep. 2, 1994, whichis a continuation-in-part application based on U.S. patent applicationSer. No. 08/144,775, filed Oct. 29, 1993 and PCT WO 95/11992; the"encoded synthetic library (ESL)" system described in U.S. patentapplication Ser. No. 08/146,886, filed Nov. 12, 1993 which is acontinuation in part application of U.S. patent application Ser. No.07/946,239, filed Sep. 16, 1992, which is a continuation in partapplication of U.S. patent application Ser. No. 07/762,522, filed Sep.18, 1991; and the "very large scale immobilized polymer synthesis"system described in U.S. Pat. No. 5,143,854; PCT Patent Publication No.90/15070, published Dec. 13, 1990; U.S. patent application Ser. No.07/624,120, filed Dec. 6, 1990; Fodor, et al., Science, 251:767-773(2/1991); Dower, et al., Ann. Rep. Med. Chem., 26:271-180 (1991); andU.S. patent application Ser. No. 805,727, filed Dec. 6, 1991.

Using the procedures described above, random peptides were generallydesigned to have a defined number of amino acid residues in length(e.g., 12). To generate the collection of oligonucleotides encoding therandom peptides, the codon motif (NNK)x, where N is nucleotide A, C, G,or T (equimolar; depending on the methodology employed, othernucleotides can be employed), K is G or T (equimolar), and x is aninteger corresponding to the number of amino acids in the peptide (e.g.,12) was used to specify any one of the 32 possible codons resulting fromthe NNK motif: 1 for each of 12 amino acids, 2 for each of 5 aminoacids, 3 for each of 3 amino acids, and only one of the three stopcodons. Thus, the NNK motif encodes all of the amino acids, encodes onlyone stop codon, and reduces codon bias.

In the systems employed, the random peptides were presented either onthe surface of a phage particle, as part of a fusion protein comprisingeither the pIII or the pVIII coat protein of a phage fd derivative(peptides on phage) or as a fusion protein with the LacI peptide fusionprotein bound to a plasmid (peptides on plasmids).

The phage or plasmids, including the DNA encoding the peptides, wereidentified and isolated by an affinity enrichment process usingimmobilized TPO-R. The affinity enrichment process, sometimes called"panning," involves multiple rounds of incubating the phage, plasmids,or polysomes with the immobilized receptor, collecting the phage,plasmids, or polysomes that bind to the receptor (along with theaccompanying DNA or mRNA), and producing more of the phage or plasmids(along with the accompanying LacI-peptide fusion protein) collected. Theextracellular domain (ECD) of the TPO-R typically was used duringpanning.

After several rounds of affinity enrichment, the phage or plasmids andaccompanying peptides were examined by ELISA to determine if thepeptides bind specifically to TPO-R. This assay was carried outsimilarly to the procedures used in the affinity enrichment process,except that after removing unbound phage, the wells were typicallytreated with rabbit anti-phage antibody, then with alkaline phosphatase(AP)-conjugated goat anti-rabbit antibody. The amount of alkalinephosphatase in each well was determined by standard methods. A similarELISA procedure for use in the peptides on plasmids system is describedin detail below.

By comparing test wells with control wells (no receptor), one candetermine whether the fusion proteins bind to the receptor specifically.The phage pools found to bind to TPO-R were screened in a colony liftprobing format using radiolabelled monovalent receptor. This probe canbe produced using protein kinase A to phosphorylate a peptide sequencefused to the C-terminus of the soluble receptor. The "engineered" formof the TPO receptor is then expressed in host cells, typically CHOcells. Following PI-PLC harvest of the receptors, the receptor wastested for binding to TPO or TPO-R specific phage clones. The receptoris then labeled to high specific activity with ³³ P for use as amonovalent probe to identify high affinity ligands using colony lifts.

Peptides found to bind specifically to the receptor were thensynthesized as the free peptide (e.g., no phage) and tested in ablocking assay. The blocking assay was carried out in similar fashion tothe ELISA, except that TPO or a reference peptide was added to the wellsbefore the fusion protein (the control wells were of two types: (1) noreceptor; and (2) no TPO or reference peptide). Fusion proteins forwhich the binding to the receptor was blocked by TPO or the referencepeptide contain peptides in the random peptide portion that arepreferred compounds of the invention.

TPO-R, as well as its extracellular domain, were produced in recombinanthost cells. One useful form of TPO-R is constructed by expressing theprotein as a soluble protein in baculovirus transformed host cells usingstandard methods; another useful form is constructed with a signalpeptide for protein secretion and for glycophospholipid membrane anchorattachment. This form of anchor attachment is called "PIG-tailing". SeeCaras, et al., Science, 243:1196-1198 (1989) and Lin, et al., Science,249:677-679 (1990).

Using the PIG-tailing system, one can cleave the receptor from thesurface of the cells expressing the receptor (e.g., transformed CHOcells selected for high level expression of receptor with a cell sorter)with phospholipase C. The cleaved receptor still comprises a carboxyterminal sequence of amino acids, called the "HPAP tail", from thesignal protein for membrane attachment and can be immobilized withoutfurther purification. The recombinant receptor protein can beimmobilized by coating the wells of microtiter plates with an anti-HPAPtail antibody (Ab 179 or MAb 179), blocking non-specific binding withbovine serum albumin (BSA) in PBS, and then binding cleaved recombinantreceptor to the antibody. Using this procedure, one should perform theimmobilization reaction in varying concentrations of receptor todetermine the optimum amount for a given preparation, because differentpreparations of recombinant protein often contain different amounts ofthe desired protein. In addition, one should ensure that theimmobilizing antibody is completely blocked (with TPO or some otherblocking compound) during the affinity enrichment process. Otherwise,unblocked antibody can bind undesired phage during the affinityenrichment procedure. One can use peptides that bind to the immobilizingantibody to block unbound sites that remain after receptorimmobilization to avoid this problem or one can simply immobilize thereceptor directly to the wells of microtiter plates, without the aid ofan immobilizing antibody. See U.S. patent application Ser. No.07/947,339, filed Sep. 18, 1992, incorporated herein by reference.

When using random peptide generation systems that allow for multivalentligand-receptor interaction, one must recognize that the density of theimmobilized receptor is an important factor in determining the affinityof the ligands that can bind to the immobilized receptor. At higherreceptor densities (e.g., each anti-receptor antibody-coated welltreated with 0.25 to 0.5 mg of receptor), multivalent binding is morelikely to occur than at lower receptor densities (e.g., eachanti-receptor antibody-coated well treated with 0.5 to 1 ng of thereceptor). If multivalent binding is occurring, then one will be morelikely to isolate ligands with relatively lower affinity, unless oneuses high densities of immobilized receptor to identify lead compoundsand uses lower receptor densities to isolate higher affinity derivativecompounds.

To discriminate among higher affinity peptides, a monovalent receptorprobe frequently is used. This probe can be produced using proteinkinase A to phosphorylate a peptide sequence fused to the C-terminus ofthe soluble receptor. The "engineered" form of the TPO receptor is thenexpressed in host cells, typically CHO cells. Following PI-PLC harvestof the receptors, the receptor was tested for binding to TPO or TPO-Rspecific phage clones. The receptor is then labeled to high specificactivity with ³³ P for use as a monovalent probe to identify highaffinity ligands using colony lifts.

Preferred screening methods to facilitate identification of peptideswhich bind TPO-R involve first identifying lead peptides which bind tothe extracellular domain of the receptor and then making other peptideswhich resemble the lead peptides. Specifically, using a pIII orpVIII-based peptides on phage system, a random library can be screenedto discover a phage that presents a peptide that binds to TPO-R. Thephage DNAs are sequenced to determine the sequences of the peptidesdisplayed on the surface of the phages.

Clones capable of specific binding to the TPO-R were identified from arandom linear 10-mer pVIII library and a random cyclic 10-mer and 12-merpVIII libraries. The sequences of these peptides serve as the basis forthe construction of other peptide libraries designed to contain a highfrequency of derivatives of the initially identified peptides. Theselibraries can be synthesized so as to favor the production of peptidesthat differ from the binding peptide in only a few residues. Thisapproach involves the synthesis of an oligonucleotide with the bindingpeptide coding sequence, except that rather than using pure preparationsof each of the four nucleoside triphosphates in the synthesis, one usesmixtures of the four nucleoside triphosphates (i.e., 55% of the"correct" nucleotide, and 15% each of the other three nucleotides is onepreferred mixture for this purpose and 70% of the "correct" nucleotideand 10% of each of the other three nucleotides is another preferredmixture for this purpose) so as to generate derivatives of the bindingpeptide coding sequence.

A variety of strategies were used to derivatize the lead peptides bymaking "mutagenesis on a theme" libraries. These included a pVIIIphagemid mutagenesis library based on the consensus sequence mutagenizedat 70:10:10:10 frequency and extended on each terminus with randomresidues to produce clones which include the sequence (SEQ ID NO: 21)XXXX (C, S, P, or R) TLREWL XXXXXX (C or S). A similarextended/mutagenized library was constructed using thepeptides-on-plasmids system to produce clones which include the sequence(SEQ ID NO: 22) XXXX (C, S, P, or R) TLREWL XXXXXXX. An additionalextended/mutagenized library (SEQ ID NO: 23), XXXX (C, S, P, or R)TLREWL XXXXXX (C or S), was constructed using the polysome displaysystem. All three libraries were screened with peptide elution andprobed with radiolabeled monovalent receptor.

The "peptides on plasmids" techniques was also used for peptidescreening and mutagenesis studies and is described in greater detail inU.S. Pat. No. 5,338,665, which is incorporated herein by reference forall purposes. According to this approach, random peptides are fused atthe C-terminus of LacI through expression from a plasmid vector carryingthe fusion gene. Linkage of the LacI-peptide fusion to its encoding DNAoccurs via the lacO sequences on the plasmid, forming a stablepeptide-LacI-plasmid complex that can be screened by affinitypurification (panning) on an immobilized receptor. The plasmids thusisolated can then be reintroduced into E. coli by electroporation toamplify the selected population for additional rounds of screening, orfor the examination of individual clones.

In addition, random peptide screening and mutagenesis studies wereperformed using a modified C-terminal Lac-I display system in whichdisplay valency was reduced ("headpiece dimer" display system). Thelibraries were screened and the resulting DNA inserts were cloned as apool into a maltose binding protein (MBP) vector allowing theirexpression as a C-terminal fusion protein. Crude cell lysates fromrandomly picked individual MBP fusion clones were then assayed for TPO-Rbinding in an ELISA format, as discussed above.

Peptide mutagenesis studies were also conducted using the polysomedisplay system, as described in co-pending application U.S. patentapplication Ser. No. 08/300,262, filed Sep. 2, 1994, which is acontinuation-in-part application based on U.S. patent application Ser.No. 08/144,775, filed Oct. 29, 1993 and PCT WO 95/11992, each of whichis incorporated herein by references for all purposes. A mutagenesislibrary was constructed based on the sequence (SEQ ID NO: 24) X X X X(C,P,R,or S) t l r e f l X X X X X X (C or S), in which X represents arandom NNK codon, and the lower case letters represent amino acid codonscontaining 70:10:10:10 mutagenesis at positions 1 and 2 and K (G or T)at position 3 of the codon. The library was panned for 5 rounds againstTPO receptor which had been immobilized on magnetic beads. After thefifth round, the PCR amplified pool was cloned into pAFF6 and the ELISApositive clones were sequenced. The sequences were subcloned into an MBPvector and their binding affinities were determined by an MBP ELISA.

To immobilize the TPO-R for polysome screening, Ab 179 was firstchemically conjugated to tosyl-activated magnetic beads (available fromDynal Corporation) as described by the manufacturer. The beads wereincubated with antibody in 0.5M borate buffer (pH 9.5) overnight at roomtemperature. The beads were washed and combined with TPO-R containingthe "HPAP" tail. The antibody coated beads and receptor were incubatedfor 1 hour at 4° C., and the beads were washed again prior to adding thepolysome library.

Screening of the various libraries described above yielded the TPOreceptor binding peptides shown in Tables 1 and 2 below, as well asothers not listed herein. The peptides are set forth herein startingfrom the N-terminus to the C-terminus. Such peptides typically have NH₂-- as the amino terminal and --COOH as the acid terminal. Alternatively,however, such peptides can have NH₂ -- as the amino termial and --CONH₂as the acid terminal, or X-- as the amino terminal and --COOH as theacid terminal, wherein X is, for example, Ac, Z, Boc, etc.

                                      TABLE 1    __________________________________________________________________________    (SEQ ID NOS. 25-58, respectively)    Peptide    __________________________________________________________________________    R E G P T L R Q W M    R E G P T L R Q W M    S R G M T L R E W L    E G P T L R G W L A    R E G Q T L K E W L    E R G P F W A K A C    R E G P R C V M W M    C S G L T L R E W L V C    C L T G P F V T Q W L Y E C    C G E G L T L T Q W L E H C    C R A G P T L L E W L T L C    C R A G P T L L E W L T L C    C R Q G P T L T A W L L E C    C A D G P T L R E W I S F C    C E L V G P S L M S W L T C    C G T E G P T L S T W L D C    C D Q L G V T L S R W L E C    S G T G L T L R E W L G S F S L L S    C P E G P T L L Q W L K R G Y S S C    R G D G P T L S Q W L Y S L M I M C    M V A G P T L R E F I A S L P I H C    S M Q G P T F R E W V S M M K V L C    S V Q C G P T L R Q W L A A R N H L S    G N A D G P T L R Q W L E G R R P K N    S V R C G P T L R Q W L A A R T H L S    L A I E G P T L R Q W L H G N G R D T    H G R V G P T L R E W K T Q V A T K K    C A D G P T L R E W I S F C    I S D G P T L K E W L S V T R G A S    S I E G P T L R E W L T S R T P H S    T I K G P T L R Q W L K S R E H T S    G N A D G P T L R Q W L E G R R P K N    S I E G P T L R E W L T S R T P H S    I S D G P T L K E W L S V T R G A S    __________________________________________________________________________

                                      TABLE 2    __________________________________________________________________________    (SEQ. ID NOS. 59-167, respectively)    Peptide    __________________________________________________________________________    C S L E D L R K R C    C R R S E L L E R C    C T F K Q F L D G C    C T R G E W L R C C    C T L R Q W L Q G C    C T L E E L R A C C    C T R E E L M R L C    C Q R A D L I N F C    C N R N D L L L F C    C T R T E W L H G C    C T L E F M N G C    C S L G E L R R L C    C N I N Q L R S I C    C T M R Q F L V C C    C T R S E W L E R C    C T L H E Y L S G C    C T R E E L L R Q C    C T F R E F V N G C    C S R A D F L A A C    C S C A Q V V Q C C    C T L R Q W I L L G M C    C T L R E W L H G G F C    C T L R A W L M S E T C    C T L R A W L M E S C C    C T F Q V W K L A R N C    C L L R E W L D X R T C    C V L R E W L L X X S C    C L L S E F L A G Q Q C    C S L R Q Y L D F G L G S C    C T L Q E L K Q S S L Y E C    C D L S E L K T H G Y A Y C    C K L S D W L M N G V A A C    C S L Q E F L S H G G Y V C    C S L K E F L H S G L M Q C    C T F R Q L L E Y G V S S C    C T M R E F L V A S G V A C    C T L A E F L A S G V E Q C    C T L A E F L A S G V E Q C    C T L K E W L V S H E V W C    C T L R E F L S L G M N A C    C T L R E F L D P T T A V C    C S L L E F L A L G V A L C    G G G R G C T L K Q W K Q G D C G R S    C N R S Q L L A A C    C T L Q Q W L S G C    C T L R E F K A G C    C T R A Q F L K G C    C T L R E F N R G C    C T L S D F K R G C    C T F R Q W K E A C    C T L S E F R G G C    C T L Q E F L E G C    C T L Q Q W K D G C    C T R S Q W L E G C    C S L Q E F K H G C    C T L G E W K R G C    C T L W G C G K R G C    C T L Q E W R G G C    C T R L S G C W L C    C T R T Q W L L D C    C T L A E F R R G C    C T S T Q W L L A C    C S R S Q F L R S C    C T L R E W L E G C    C T L R E F L L M G A C    C T L K E W L L W S S C    C T L L E W L R N P V C    C T L R Q W L G D A W C    C T L G Q W L Q M G M C    C T L R E W V F A G L C    C L L L E F L S G A D C    C T L G E F L A G H L C    C R L R E F L V D L T C    C S F R S W L V D Q T C    C T L R E W L E D L G C    C T L Q D W L V S W T C    C T L S E W L S E L S C    C T L M Q W L G G W P C    C T L R E W L S Y G T C    C T L Q E W L S G G L C    G S H G C T L R E W L C M K I V P C    Q W Q G C T L R D C I L R G V F W S    S V N S C T L R E F L T G C R V F C    S Y D G C T L R H W L M D I Y G D C    Q R S G C T L R D W V L L N C L A S    N Y R G C T L S Q W V S E Q I V G C    G R S G C T L R E Y L G G M C Y L S    A S W Y C T V P E L M E M Q L P E C    G S T G C T L R E X L H M L G L D C    A C E G C T L R Q W L E Y V R V G C    A Q R G C T L Q Y F V S Y G X D M C    G V C G C T L R E F L A I P H T S C    S E G G C T L R E W V A S S L A N C    S N S R C T L R E W I I Q G C D F S    S N S R C T L R E W I I Q G C D F S    C L G C T L S Q W R K R T R C D T H    Y R G C S R A Q L L G G E C R K K    G R G C T L K Q W K Q G D C G R S    V R G G C A L R D W V A G E C F D W T    L W R G C T L N G F K S R H C G S P E    C T L R S W K H R G C A P    G R G C T R A Q W L A G C C T G H    R A G C T L R E F R K G C L A L    K R G C T L A E M I R G C N R S N    G R G C T L K Q W K Q G D C G R S    R W R G C S L A K L K K G A A C G R G    R G G C T L R E W R R V R V I N    G R G C T L K Q W K Q G D C G R S    R Y G C T R H Q W L V G T C V R H    __________________________________________________________________________

IC₅₀ values for some additional representative peptides are given in thetable below. A variety of methods can be used to evaluate IC₅₀ values.For example, an equilibrium binding ELISA assay, using either MBP-TPO orlacI-peptide tracer, was used to determine whether the peptides inhibitthe binding of TPO to the extracellular domain of the TPO receptor.Typically, the IC₅₀ value were determined using the free peptide. TheIC₅₀ value can be determined using the free peptide, which optionallycan be C-terminally amidated, or can be prepared as an ester or othercarboxy amide.

To recreate the exact sequence displayed on the phage, the N-terminaland C-terminal amino acids of the synthetic peptides are often precededby one or two glycine residues. These glycines are not believed to benecessary for binding or activity. Likewise, to mimic the exact sequenceof peptides displayed on polysomes, the C-terminal amino acids of thesynthetic peptides are often preceded by the sequence M A S. Again, thissequence is not believed to be necessary for binding or activity.

IC₅₀ values are indicated symbolically by the symbols "-", "+", and"++". For examples, those peptides which showed IC₅₀ values in excess of200 μM are indicated with a "-". Those peptides which gave IC₅₀ valuesof less than or equal to 200 μM are given a "+", while those which gaveIC₅₀ values of 500 nm or less are indicated with a "++". Those peptideswhich gave IC₅₀ values at or near the cutoff point for a particularsymbol are indicated with a hybrid designator, e.g., "±". Those peptidesfor which IC₅₀ values were not determined are listed as "N.D.". The IC₅₀value for peptides having the structure: (SEQ ID NO: 15) G G C T L R E WL H G G F C G G was 500 nm or less. (Note the N-terminal and C-terminalamino acids were preceded by two glycines to recreate the exact sequencedisplayed by the phage. These glycines are not believed to be necessaryfor binding or activity.)

                                      TABLE 3    __________________________________________________________________________    (SEQ ID NOS 6, 7, 8, 9, 168, 11 & 10, respectively)    Peptide                               Affinity    __________________________________________________________________________    G G C A D G P T L R E W I S F C G G   ++    G N A D G P T L R Q W L E G R R P K N  ++    G G C A D G P T L R E W I S F C G G K  ++    T I K G P T L R Q W L K S R E H T S    ++    G P T L R Q W L                        -    L A I E G P T L R Q W L H G N G R D T  ++    S I E G P T L R E W L T S R T P H S    ++    __________________________________________________________________________

The tables above, especially Table 3, illustrate that a preferred corepeptide comprises a sequence of amino acids: (SEQ ID NO:2)

    X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6 X.sub.7

where X₁ is C, L, M, P, Q, V; X₂ is F, K, L, N, Q, R, S, T or V; X₃ isC, F, I, L, M, R, S, V or W; X₄ is any of the 20 genetically codedL-amino acids; X₅ is A, D, E, G, K, M, Q, R, S, T, V or Y; X₆ is C, F,G, L, M, S, V, W or Y; and X₇ is C, G, I, K, L, M, N, R or V.

In a preferred embodiment the core peptide comprises a sequence of aminoacids: (SEQ ID NO:3)

    X.sub.8 G X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 W X.sub.7

where X₁ is L, M, P, Q, or V; X₂ is F, R, S, or T; X₃ is F, L, V, or W;X₄ is A, K, L, M, R, S, V, or T; X₅ is A, E, G, K, M, Q, R, S, or T; X₇is C, I, K, L, M or V; and each X₈ residue is independently selectedfrom any of the 20 genetically coded L-amino acids, their stereoisomericD-amino acids; and non-natural amino acids. Preferably, each X₈ residueis independently selected from any of the 20 genetically coded L-aminoacids and their stereoisomeric D-amino acids. In a preferred embodiment(SEQ ID NO:4), X₁ is P; X₂ is T; X₃ is L; X₄ is R; X₅ is E or Q; and X₇is I or L.

More preferably, the core peptide comprises a sequence of amino acids:(SEQ ID NO:5)

    X.sub.9 X.sub.8 G X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 W X.sub.7

where X₉ is A, C, E, G, I, L , M, P, R, Q, S, T, or V; and X₈ is A, C,D, E, K, L, Q, R, S, T, or V. More preferably, X₉ is A or I; and X₈ isD, E, or K.

Particularly preferred peptides include: (SEQ ID NOS 6-13, respectively)G G C A D G P T L R E W I S F C G G; G N A D G P T L R Q W L E G R R P KN; G G C A D G P T L R E W I S F C G G K; T I K G P T L R Q W L K S R EH T S; S I E G P T L R E W L T S R T P H S; L A I E G P T L R Q W L H GN G R D T; C A D G P T L R E W I S F C; and I E G P T L R Q W L A A R A.

In further embodiments of the invention, preferred peptides for use inthis invention include peptides having a core structure comprisingsequence of amino acids: sequence of amino acids: (SEQ ID NO:14)

    C X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6 X.sub.7

where X₂ is F, K, L, N, Q, R, S, T or V; X₃ is C, F, I, L, M, R, S or V;X₄ is any of the 20 genetically coded L-amino acids; X₅ is A, D, E, G,S, V or Y; X₆ is C, F, G, L, M, S, V, W or Y; and X₇ is C, G, I, K, L,M, N, R or V. In a more preferred embodiment, X₄ is A, E, G, H, K, L, M,P, Q, R, S, T, or W. In a further embodiment, X₂ is S or T; X₃ is L orR; X₄ is R; X₅ is D, E, or G; X₆ is F, L, or W; and X₇ is I, K, L, R, orV. Particularly preferred peptides include: (SEQ ID NO:15) G G C T L R EW L H G G F C G G.

In a further embodiment, preferred peptides for use in this inventioninclude peptides having a structure comprising a sequence of aminoacids: (SEQ ID NO: 16)

    X.sub.8 C X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6 X.sub.7

where X₂ is F, K, L, N, Q, R, S, T or V; X₃ is C, F, I, L, M, R, S, V orW; X₄ is any of the 20 genetically coded L-amino acids; X₅ is A, D, E,G, K, M, Q, R, S, T, V or Y; X₆ is C, F, G, L, M, S, V, W or Y; X₇ is C,G, I, K, L, M, N, R or V; and X₈ is any of the 20 genetically codedL-amino acids. In some embodiments, X₈ is preferably G, S, Y, or R.

Peptides and peptidomimetics having an IC₅₀ of greater than about 100 mMlack sufficient binding to permit use in either the diagnostic ortherapeutic aspects of this invention. Preferably, for diagnosticpurposes, the peptides and peptidomimetics have an IC₅₀ of about 2 mM orless and, for pharmaceutical purposes, the peptides and peptidomimeticshave an IC₅₀ of about 100 μM or less.

The binding peptide sequence also provides a means to determine theminimum size of a TPOR binding compound of the invention. Using the"encoded synthetic library" (ESL) system or the "very large scaleimmobilized polymer synthesis" system, one can not only determine theminimum size of a peptide with such activity, but one can also make allof the peptides that form the group of peptides that differ from thepreferred motif (or the minimum size of that motif) in one, two, or moreresidues. This collection of peptides can then be screened for abilityto bind to TPO-receptor. These immobilized polymers synthesis systems orother peptide synthesis methods can also be used to synthesizetruncation analogs, deletion analogs, substitution analogs, andcombinations thereof all of the peptide compounds of the invention.

The peptides and peptide mimetics of the present invention were alsoevaluated in a thrombopoietin dependent cell proliferation assay, asdescribed in greater detail in Example 2 below. Cell proliferation ismeasured by techniques known in the art, such as an MTT assay whichcorrelates with ³ H-thymidine incorporation as an indication of cellproliferation (see Mossmann, J. Immunol. Methods, 65:55 (1983)). Thepeptides tested stimulated proliferation of TPO-R transfected Ba/F3cells in a dose dependent manner as shown in FIG. 1A. These peptideshave no effect on the parental cell line as shown in FIG. 1B.

FIGS. 7 to 9 show the results of a further assay evaluating activity ofthe peptides and peptide mimetics of the invention. In this assay miceare made thrombocytopenic with carboplatin. FIG. 7 depicts typicalresults when Balb/C mice are treated with carboplatin (125 mg/kgintraperitoneally) on Day 0. The dashed lines represent untreatedanimals from three experiments. The solid line representcarboplatin-treated groups in three experiments. The heavy solid linesrepresent historical data. FIG. 8 depicts the effect of carboplatintitration on platelet counts in mice treated with the indicated amountsof carboplatin (in mg/kg, intraperitoneally (ip) on Day 0). FIG. 9depicts amelioration of carboplatin-induced thrombocytopenia on Day 10by peptide AF12513 (513). Carboplatin (CBP; 50-125 mg/kg,intraperitoneally) was administered on Day 0. AF12513 (1 mg/kg, ip) wasgiven on Days 1-9. These results show the peptides of the invention canameliorate thrombocytopenia in a mouse model.

In addition, certain peptides of the present invention can be dimerizedor oligomerized, thereby increasing the affinity and/or activity of thecompounds. To investigate the effect that peptidedimerization/oligomerization has on TPO mimetic potency in cellproliferation assays, a C-terminally biotinylated analog of the peptide(SEQ ID NO: 6) G G C A D G P T L R E W I S F C G G was synthesized (SEQID NO: 8) (G G C A D G P T L R E W I S F C G G K (Biotin)). The peptidewas preincubated with streptavidin in serum-free HEPES-buffered RPMI ata 4:1 molar ratio. The complex was tested for stimulation of cellproliferation of TPO-R transfected Ba/F3 cells, as above, alongside freebiotinylated peptide and the unbiotinylated parental peptide. FIG. 2Ashows the results of the assay for the complexed biotinylated peptide(AF 12885 with streptavidin (SA)) for both the transfected and parentalcell lines. FIG. 2B shows the results of the assay for the freebiotinylated peptide (AF 12285) for both the transfected and parentalcell lines. FIG. 2C shows the results of the assay for streptavidinalone for both the transfected and parental cell lines. These figuresillustrate that the pre-formed complex was approximately 10 times morepotent as the free peptide.

The specificity of the binding and activity of the peptides of theinvention was also examined by studying the cross reactivity of thepeptides for the erythropoietin receptor (EPO-R). The EPO-R is also amember of the hematopoietin growth factor receptor family, as is TPO-R.The peptides of the invention, as well as TPO, EPO, and a knownEPO-binding peptide, were examined in a cell proliferation assay usingan EPO-dependent cell line. This assay utilized FDCP-1, a growth factordependent murine multi-potential primitive hematopoietic progenitor cellline (see, e.g., Dexter, et al., J. Exp. Med., 152:1036-1047 (1981)) asthe parental cell line. This cell line can proliferate, but notdifferentiate when supplemented with WEHI-3-conditioned media (a mediumthat contains IL-3, ATCC number T1B68). The parental cell line istransfected with human or murine EPO-R to produce the FDCP-1-EPO-R cellline. These transfected cell lines can proliferate, but notdifferentiate in the presence of human or murine EPO.

The cells were grown to half stationary density in the presence of thenecessary growth factors. The cells are then washed in PBS and starvedfor 16-24 hours in whole media without the growth factors. Afterdetermining the viability of the cells, stock solutions (in whole mediawithout the growth factors) are made to give about 10⁵ cells per 50microliters. Serial dilutions of the compounds (typically, the freesolution phase peptide as opposed to a phage-bound or other bound orimmobilized peptide) to be tested are made in 96-well tissue cultureplates for a final volume of 50 microliters per well. Cells (50microliters) are added to each well and the cells are incubated for24-48 hours, at which point the negative controls should die or bequiescent. Cell proliferation is then measured by techniques known inthe art, such as an MTT assay.

FIGS. 3A-G show the results of a series of control experiments showingthe activity of TPO, the peptides of the present invention, EPO, andEPO-R binding peptides in a cell proliferation assay using either theTPO-R transfected Ba/F3 cell line and its corresponding parental line,or an EPO-dependent cell line and its corresponding parental line. FIG.3A depicts the results for TPO in the cell proliferation assay using theTPO-R transfected Ba/F3 cell line and its corresponding parental line.FIG. 3B depicts the results for EPO in the cell proliferation assayusing the TPO-R transfected Ba/F3 cell line and its correspondingparental line. FIG. 3C depicts the results for complexed biotinylatedpeptide (AF 12285 with streptavidin (SA)) and a complexed form of abiotinylated EPO-R binding peptide (AF 11505 with SA) in the TPO-Rtransfected Ba/F3 cell line. The results for the corresponding parentalcell line are shown in FIG. 3D. FIG. 3E depicts the results for TPO inthe cell proliferation assay using the EPO-dependent cell line. FIG. 3Fdepicts the results for EPO in the cell proliferation assay using theEPO-dependent cell line. FIG. 3G depicts the results for complexedbiotinylated peptide (AF 12285 with streptavidin (SA)) and the complexedform of a biotinylated EPO-R binding peptide (AF 11505 with SA) in theEPO-dependent cell line. These results show that the peptides of theinvention bind and activate the TPO-R with a high degree of specificity.

IV. PREPARATION OF PEPTIDES AND PEPTIDE MIMETICS

A. SOLID PHASE SYNTHESIS

The peptides of the invention can be prepared by classical methods knownin the art, for example, by using standard solid phase techniques. Thestandard methods include exclusive solid phase synthesis, partial solidphase synthesis methods, fragment condensation, classical solutionsynthesis, and even by recombinant DNA technology. See, e.g.,Merrifield, J. Am. Chem. Soc., 85:2149 (1963), incorporated herein byreference. On solid phase, the synthesis is typically commenced from theC-terminal end of the peptide using an alpha-amino protected resin. Asuitable starting material can be prepared, for instance, by attachingthe required alpha-amino acid to a chloromethylated resin, ahydroxymethyl resin, or a benzhydrylamine resin. One suchchloromethylated resin is sold under the tradename BIO-BEADS SX-1 by BioRad Laboratories, Richmond, CA, and the preparation of the hydroxymethylresin is described by Bodonszky, et al., Chem. Ind. (London), 38:1597(1966). The benzhydrylamine (BHA) resin has been described by Pietta andMarshall, Chem. Commn., 650 (1970) and is commercially available fromBeckman Instruments, Inc., Palo Alto, Calif., in the hydrochloride form.

Thus, the compounds of the invention can be prepared by coupling analpha-amino protected amino acid to the chloromethylated resin with theaid of, for example, cesium bicarbonate catalyst, according to themethod described by Gisin, Helv. Chim. Acta., 56:1467 (1973). After theinitial coupling, the alpha-amino protecting group is removed by achoice of reagents including trifluoroacetic acid (TFA) or hydrochloricacid (HCl) solutions in organic solvents at room temperature.

The alpha-amino protecting groups are those known to be useful in theart of stepwise synthesis of peptides. Included are acyl type protectinggroups (e.g., formyl, trifluoroacetyl, acetyl), aromatic urethane typeprotecting groups (e.g. benzyloxycarboyl (Cbz) and substituted Cbz),aliphatic urethane protecting groups (e.g., t-butyloxycarbonyl (Boc),isopropyloxycarbonyl, cyclohexyloxycarbonyl) and alkyl type protectinggroups (e.g., benzyl, triphenylmethyl). Boc and Fmoc are preferredprotecting groups. The side chain protecting group remains intact duringcoupling and is not split off during the deprotection of theamino-terminus protecting group or during coupling. The side chainprotecting group must be removable upon the completion of the synthesisof the final peptide and under reaction conditions that will not alterthe target peptide.

The side chain protecting groups for Tyr include tetrahydropyranyl,tert-butyl, trityl, benzyl, Cbz, Z--Br--Cbz, and 2,5-dichlorobenzyl. Theside chain protecting groups for Asp include benzyl, 2,6-dichlorobenzyl,methyl, ethyl, and cyclohexyl. The side chain protecting groups for Thrand Ser include acetyl, benzoyl, trityl, tetrahydropyranyl, benzyl,2,6-dichlorobenzyl, and Cbz. The side chain protecting group for Thr andSer is benzyl. The side chain protecting groups for Arg include nitro,Tosyl (Tos), Cbz, adamantyloxycarbonyl mesitoylsulfonyl (Mts), or Boc.The side chain protecting groups for Lys include Cbz,2-chlorobenzyloxycarbonyl (2-Cl--Cbz), 2-bromobenzyloxycarbonyl(2-BrCbz), Tos, or Boc.

After removal of the alpha-amino protecting group, the remainingprotected amino acids are coupled stepwise in the desired order. Anexcess of each protected amino acid is generally used with anappropriate carboxyl group activator such as dicyclohexylcarbodiimide(DCC) in solution, for example, in methylene chloride (CH₂ Cl₂),dimethyl formamide (DMF) mixtures.

After the desired amino acid sequence has been completed, the desiredpeptide is decoupled from the resin support by treatment with a reagentsuch as trifluoroacetic acid or hydrogen fluoride (HF), which not onlycleaves the peptide from the resin, but also cleaves all remaining sidechain protecting groups. When the chloromethylated resin is used,hydrogen fluoride treatment results in the formation of the free peptideacids. When the benzhydrylamine resin is used, hydrogen fluoridetreatment results directly in the free peptide amide. Alternatively,when the chloromethylated resin is employed, the side chain protectedpeptide can be decoupled by treatment of the peptide resin with ammoniato give the desired side chain protected amide or with an alkylamine togive a side chain protected alkylamide or dialkylamide. Side chainprotection is then removed in the usual fashion by treatment withhydrogen fluoride to give the free amides, alkylamides, ordialkylamides.

These solid phase peptide synthesis procedures are well known in the artand further described by John Morrow Stewart and Janis Dillaha Young,Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).

Using the "encoded synthetic library" or "very large scale immobilizedpolymer synthesis" system described in U.S. patent application Ser. No.07/492,462, filed Mar. 7, 1990; Ser. No. 07/624,120, filed Dec. 6, 1990;and Ser. No. 07/805,727, filed Dec. 6, 1991; one can not only determinethe minimum size of a peptide with such activity, one can also make allof the peptides that form the group of peptides that differ from thepreferred motif (or the minimum size of that motif) in one, two, or moreresidues. This collection of peptides can then be screened for abilityto bind to TPO-R. This immobilized polymer synthesis system or otherpeptide synthesis methods can also be used to synthesize truncationanalogs and deletion analogs and combinations of truncation and deletionanalogs of all of the peptide compounds of the invention.

B. SYNTHETIC AMINO ACIDS

These procedures can also be used to synthesize peptides in which aminoacids other than the 20 naturally occurring, genetically encoded aminoacids are substituted at one, two, or more positions of any of thecompounds of the invention. For instance, naphthylalanine can besubstituted for tryptophan, facilitating synthesis. Other syntheticamino acids that can be substituted into the peptides of the presentinvention include L-hydroxypropyl, L-3, 4-dihydroxyphenylalanyl, d aminoacids such as L-d-hydroxylysyl and D-d-methylalanyl, L-α-methylalanyl, βamino acids, and isoquinolyl. D amino acids and non-naturally occurringsynthetic amino acids can also be incorporated into the peptides of thepresent invention (see, e.g., Roberts, et al., Unusual Amino/Acids inPeptide Synthesis, 5(6):341-449 (1983)).

One can replace the naturally occurring side chains of the 20genetically encoded amino acids (or D amino acids) with other sidechains, for instance with groups such as alkyl, lower alkyl, cyclic 4-,5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amide di(loweralkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivativesthereof, and with 4-, 5-, 6-, to 7-membered hetereocyclic. Inparticular, proline analogs in which the ring size of the prolineresidue is changed from 5 members to 4, 6, or 7 members can be employed.Cyclic groups can be saturated or unsaturated, and if unsaturated, canbe aromatic or non-aromatic. Heterocyclic groups preferably contain oneor more nitrogen, oxygen, and/or sulphur heteroatoms. Examples of suchgroups include the furazanyl, furyl, imidazolidinyl, imidazolyl,imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholino),oxazolyl, piperazinyl (e.g. 1-piperazinyl), piperidyl (e.g. 1-piperidyl,piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl,pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g. 1-pyrrolidinyl),pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl(e.g. thiomorpholino), and triazolyl. These heterocyclic groups can besubstituted or unsubstituted. Where a group is substituted, thesubstituent can be alkyl, alkoxy, halogen, oxygen, or substituted orunsubstituted phenyl.

One can also readily modify the peptides of the instant invention byphosphorylation (see, e.g., W. Bannwarth, et al., Biorganic andMedicinal Chemistry Letters, 6(17):2141-2146 (1996)), and other methodsfor making peptide derivatives of the compounds of the present inventionare described in Hruby, et al., Biochem. J., 268(2):249-262 (1990).Thus, the peptide compounds of the invention also serve as a basis toprepare peptide mimetics with similar biological activity.

C. TERMINAL MODIFICATIONS

Those of skill in the art recognize that a variety of techniques areavailable for constructing peptide mimetics with the same or similardesired biological activity as the corresponding peptide compound butwith more favorable activity than the peptide with respect tosolubility, stability, and susceptibility to hydrolysis and proteolysis.See, for example, Morgan, et al., Ann. Rep. Med. Chem., 24:243-252(1989). The following describes methods for preparing peptide mimeticsmodified at the N-terminal amino group, the C-terminal carboxyl group,and/or changing one or more of the amido linkages in the peptide to anon-amido linkage. It being understood that two or more suchmodifications can be coupled in one peptide mimetic structure (e.g.,modification at the C-terminal carboxyl group and inclusion of a --CH₂-carbamate linkage between two amino acids in the peptide).

1. N-TERMINAL MODIFICATIONS

The peptides typically are synthesized as the free acid but, as notedabove, could be readily prepared as the amide or ester. One can alsomodify the amino and/or carboxy terminus of the peptide compounds of theinvention to produce other compounds of the invention. Amino terminusmodifications include methylation (i.e., --NHCH₃ or --NH(CH₃)₂),acetylation, adding a benzyloxycarbonyl group, or blocking the aminoterminus with any blocking group containing a carboxylate functionalitydefined by RCOO--, where R is selected from the group consisting ofnaphthyl, acridinyl, steroidyl, and similar groups. Carboxy terminusmodifications include replacing the free acid with a carboxamide groupor forming a cyclic lactam at the carboxy terminus to introducestructural constraints.

Amino terminus modifications are as recited above and includealkylating, acetylating, adding a carbobenzoyl group, forming asuccinimide group, etc. (See, e.g., Murray, et al., Burger's MedicinalChemistry and Drug Discovery, 5th ed., Vol. 1, Manfred E. Wolf, ed.,John Wiley and Sons, Inc. (1995).) Specifically, the N-terminal aminogroup can then be reacted as follows:

(a) to form an amide group of the formula RC(O)NH-- where R is asdefined above by reaction with an acid halide e.g., RC(O)Cl! orsymmetric anhydride. Typically, the reaction can be conducted bycontacting about equimolar or excess amounts (e.g., about 5 equivalents)of an acid halide to the peptide in an inert diluent (e.g.,dichloromethane) preferably containing an excess (e.g., about 10equivalents) of a tertiary amine, such as diisopropylethylamine, toscavenge the acid generated during reaction. Reaction conditions areotherwise conventional (e.g., room temperature for 30 minutes).Alkylation of the terminal amino to provide for a lower alkylN-substitution followed by reaction with an acid halide as describedabove will provide for N-alkyl amide group of the formula RC(O)NR--;

(b) to form a succinimide group by reaction with succinic anhydride. Asbefore, an approximately equimolar amount or an excess of succinicanhydride (e.g., about 5 equivalents) can be employed and the aminogroup is converted to the succinimide by methods well known in the artincluding the use of an excess (e.g., ten equivalents) of a tertiaryamine such as diisopropylethylamine in a suitable inert solvent (e.g.,dichloromethane). See, for example, Wollenberg, et al., U.S. Pat. No.4,612,132 which is incorporated herein by reference in its entirety. Itis understood that the succinic group can be substituted with, forexample, C₂ -C₆ alkyl or --SR substituents which are prepared in aconventional manner to provide for substituted succinimide at theN-terminus of the peptide. Such alkyl substituents are prepared byreaction of a lower olefin (C₂ -C₆) with maleic anhydride in the mannerdescribed by Wollenberg, et al., supra and --SR substituents areprepared by reaction of RSH with maleic anhydride where R is as definedabove;

(c) to form a benzyloxycarbonyl-NH-- or a substitutedbenzyloxycarbonyl-NH-- group by reaction with approximately anequivalent amount or an excess of CBZ--Cl (i.e., benzyloxycarbonylchloride) or a substituted CBZ--Cl in a suitable inert diluent (e.g.,dichloromethane) preferably containing a tertiary amine to scavenge theacid generated during the reaction;

(d) to form a sulfonamide group by reaction with an equivalent amount oran excess (e.g., 5 equivalents) of R--S(O)₂ Cl in a suitable inertdiluent (dichloromethane) to convert the terminal amine into asulfonamide where R is as defined above. Preferably, the inert diluentcontains excess tertiary amine (e.g., ten equivalents) such asdiisopropylethylamine, to scavenge the acid generated during reaction.Reaction conditions are otherwise conventional (e.g., room temperaturefor 30 minutes);

(e) to form a carbamate group by reaction with an equivalent amount oran excess (e.g., 5 equivalents) of R--OC(O)Cl or R--OC(O)OC₆ H₄ -p-NO₂in a suitable inert diluent (e.g., dichloromethane) to convert theterminal amine into a carbamate where R is as defined above. Preferably,the inert diluent contains an excess (e.g., about 10 equivalents) of atertiary amine, such as diisopropylethylamine, to scavenge any acidgenerated during reaction. Reaction conditions are otherwiseconventional (e.g., room temperature for 30 minutes); and

(f) to form a urea group by reaction with an equivalent amount or anexcess (e.g., 5 equivalents) of R--N═C═O in a suitable inert diluent(e.g., dichloromethane) to convert the terminal amine into a urea (i.e.,RNHC(O)NH--) group where R is as defined above. Preferably, the inertdiluent contains an excess (e.g., about 10 equivalents) of a tertiaryamine, such as diisopropylethylamine. Reaction conditions are otherwiseconventional (e.g., room temperature for about 30 minutes).

2. C-TERMINAL MODIFICATIONS

In preparing peptide mimetics wherein the C-terminal carboxyl group isreplaced by an ester (i.e., --C(O)OR where R is as defined above), theresins used to prepare the peptide acids are employed, and the sidechain protected peptide is cleaved with base and the appropriatealcohol, e.g., methanol. Side chain protecting groups are then removedin the usual fashion by treatment with hydrogen fluoride to obtain thedesired ester.

In preparing peptide mimetics wherein the C-terminal carboxyl group isreplaced by the amide --C(O)NR³ R⁴, a benzhydrylamine resin is used asthe solid support for peptide synthesis. Upon completion of thesynthesis, hydrogen fluoride treatment to release the peptide from thesupport results directly in the free peptide amide (i.e., the C-terminusis --C(O)NH₂). Alternatively, use of the chloromethylated resin duringpeptide synthesis coupled with reaction with ammonia to cleave the sidechain protected peptide from the support yields the free peptide amideand reaction with an alkylamine or a dialkylamine yields a side chainprotected alkylamide or dialkylamide (i.e., the C-terminus is --C(O)NRR¹where R and R¹ are as defined above). Side chain protection is thenremoved in the usual fashion by treatment with hydrogen fluoride to givethe free amides, alkylamides, or dialkylamides.

In another alternative embodiment, the C-terminal carboxyl group or aC-terminal ester can be induced to cyclize by internal displacement ofthe --OH or the ester (--OR) of the carboxyl group or ester respectivelywith the N-terminal amino group to form a cyclic peptide. For example,after synthesis and cleavage to give the peptide acid, the free acid isconverted to an activated ester by an appropriate carboxyl groupactivator such as dicyclohexylcarbodiimide (DCC) in solution, forexample, in methylene chloride (CH₂ Cl₂), dimethyl formamide (DMF)mixtures. The cyclic peptide is then formed by internal displacement ofthe activated ester with the N-terminal amine. Internal cyclization asopposed to polymerization can be enhanced by use of very dilutesolutions. Such methods are well known in the art.

One can also cyclize the peptides of the invention, or incorporate adesamino or descarboxy residue at the terminii of the peptide, so thatthere is no terminal amino or carboxyl group, to decrease susceptibilityto proteases or to restrict the conformation of the peptide. C-terminalfunctional groups of the compounds of the present invention includeamide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy,and carboxy, and the lower ester derivatives thereof, and thepharmaceutically acceptable salts thereof.

In addition to the foregoing N-terminal and C-terminal modifications,the peptide compounds of the invention, including peptidomimetics, canadvantageously be modified with or covalently coupled to one or more ofa variety of hydrophilic polymers. It has been found that when thepeptide compounds are derivatized with a hydrophilic polymer, theirsolubility and circulation half-lives are increased and theirimmunogenicity is masked. Quite surprisingly, the foregoing can beaccomplished with little, if any, diminishment in their bindingactivity. Nonproteinaceous polymers suitable for use in accordance withthe present invention include, but are not limited to, polyalkylethersas exemplified by polyethylene glycol and polypropylene glycol,polylactic acid, polyglycolic acid, polyoxyalkenes, polyvinylalcohol,polyvinylpyrrolidone, cellulose and cellulose derivatives, dextran anddextran derivatives, etc. Generally, such hydrophilic polymers have anaverage molecular weight ranging from about 500 to about 100,000daltons, more preferably from about 2,000 to about 40,000 daltons and,even more preferably, from about 5,000 to about 20,000 daltons. Inpreferred embodiments, such hydrophilic polymers have an averagemolecular weights of about 5,000 daltons, 10,000 daltons and 20,000daltons.

The peptide compounds of the invention can be derivatized with orcoupled to such polymers using any of the methods set forth inZallipsky, S., Bioconjugate Chem., 6:150-165 (1995); Monfardini, C, etal., Bioconjugate Chem., 6:62-69 (1995); U.S. Pat. No. 4,640,835; U.S.Pat. No. 4,496,689; U.S. Pat. No. 4,301,144; U.S. Pat. No. 4,670,417;U.S. Pat. No. 4,791,192; U.S. Pat. No. 4,179,337 or WO 95/34326, all ofwhich are incorporated by reference in their entirety herein.

In a presently preferred embodiment, the peptide compounds of thepresent invention are derivatized with polyethylene glycol (PEG). PEG isa linear, water-soluble polymer of ethylene oxide repeating units withtwo terminal hydroxyl groups. PEGs are classified by their molecularweights which typically range from about 500 daltons to about 40,000daltons. In a presently preferred embodiment, the PEGs employed havemolecular weights ranging from 5,000 daltons to about 20,000 daltons.PEGs coupled to the peptide compounds of the present invention can beeither branched or unbranched. (See, e.g., Monfardini, C., et al.,Bioconjugate Chem., 6:62-69 (1995)). PEGs are commercially availablefrom Shearwater Polymers, Inc. (Huntsville, Ala.), Sigma Chemical Co.and other companies. Such PEGs include, but are not limited to,monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidylsuccinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine(MePEG-NH₂), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), andmonomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

Briefly, in one exemplar embodiment, the hydrophilic polymer which isemployed, e.g., PEG, is preferably capped at one end by an unreactivegroup such as a methoxy or ethoxy group. Thereafter, the polymer isactivated at the other end by reaction with a suitable activating agent,such as cyanuric halides (e.g., cyanuric chloride, bromide or fluoride),diimadozle, an anhydride reagent (e.g., a dihalosuccinic anhydride, suchas dibromosuccinic anhydride), acyl azide, p-diazoiumbenzyl ether,3-(p-diazoniumphenoxy)-2-hydroxypropylether) and the like. The activatedpolymer is then reacted with a peptide compound of the present inventionto produce a peptide compound derivatized with a polymer. Alternatively,a functional group in the peptide compounds of the invention can beactivated for reaction with the polymer, or the two groups can be joinedin a concerted coupling reaction using known coupling methods. FIGS.15-17 illustrate exemplar reactions schemes for derivatizing the peptidecompounds of the invention with, for example, polyethylene glycol (PEG).It will be readily appreciated that the peptide compounds of theinvention can be derivatized with PEG using a myriad of other reactionschemes known to and used by those of skill in the art.

In addition to derivatizing the peptide compounds of this invention witha hydrophilic polymer (e.g., PEG), it has now been discovered that othersmall peptides, e.g., other peptides or ligands that bind to a receptor,can also be derivatized with such hydrophilic polymers with little, ifany, loss in biological activity (e.g., binding activity, agonistactivity, antagonist activity, etc.). It has been found that when thesesmall peptides are derivatized with a hydrophlilic polymer, theirsolubility and circulation half-lives are increased and theirimmunogenicity is decreased. Again, quite surprisingly, the foregoingcan be accomplished with little, if any, loss in biological activity. Infat, in preferred embodiments, the derivatized peptides have an activitythat is 0.1 to 0.01-fold that of the unmodified peptides. In morepreferred embodiments, the derivatized peptides have an activity that is0.1 to 1-fold that of the unmodified peptides. In even more preferredembodiments, the derivatized peptides have an activity that is greaterthan the unmodified peptides.

Peptides suitable for use in this embodiment generally include thosepeptides, i.e., ligands, that bind to a receptor, such as the TPO, EPO,IL-1, G-CSF and IL-5 receptors; the hematopoietic growth factorreceptors; the cytokine receptors; the G-protein-linked receptors; thecell surface receptors, etc. Such peptides typically comprise about 150amino acid residues or less and, more preferably, about 100 amino acidresidues or less (e.g., ˜10-12 kDa). Hydrophilic polymers suitable foruse in the present invention include, but are not limited to,polyalkylethers as exemplified by polyethylene glycol and polypropyleneglycol, polylactic acid, polyglycolic acid, polyoxyalkenes,polyvinylalcohol, polyvinylpyrrolidone, cellulose and cellulosederivatives, dextran and dextran derivatives, etc. Generally, suchhydrophilic polymers have an average molecular weight ranging from about500 to about 100,000 daltons, more preferably from about 2,000 to about40,000 daltons and, even more preferably, from about 5,000 to about20,000 daltons. In preferred embodiments, such hydrophilic polymers havean average molecular weights of about 5,000 daltons, 10,000 daltons and20,000 daltons. The peptide compounds of this invention can bederivatized with using the methods described above and in the citedreferences.

D. BACKBONE MODIFICATIONS

Other methods for making peptide derivatives of the compounds of thepresent invention are described in Hruby, et al., Biochem J.,268(2):249-262 (1990), incorporated herein by reference. Thus, thepeptide compounds of the invention also serve as structural models fornon-peptidic compounds with similar biological activity. Those of skillin the art recognize that a variety of techniques are available forconstructing compounds with the same or similar desired biologicalactivity as the lead peptide compound but with more favorable activitythan the lead with respect to solubility, stability, and susceptibilityto hydrolysis and proteolysis. See Morgan, et al., Ann. Rep. Med. Chem.,24:243-252 (1989), incorporated herein by reference. These techniquesinclude replacing the peptide backbone with a backbone composed ofphosphonates, amidates, carbamates, sulfonamides, secondary amines, andN-methylamino acids.

Peptide mimetics wherein one or more of the peptidyl linkages--C(O)NH--! have been replaced by such linkages as a --CH₂ -carbamatelinkage, a phosphonate linkage, a --CH₂ -sulfonamide linkage, a urealinkage, a secondary amine (--CH₂ NH--) linkage, and an alkylatedpeptidyl linkage --C(O)NR⁶ -- where R⁶ is lower alkyl! are preparedduring conventional peptide synthesis by merely substituting a suitablyprotected amino acid analogue for the amino acid reagent at theappropriate point during synthesis.

Suitable reagents include, for example, amino acid analogues wherein thecarboxyl group of the amino acid has been replaced with a moietysuitable for forming one of the above linkages. For example, if onedesires to replace a --C(O)NR-- linkage in the peptide with a --CH₂-carbamate linkage (--CH₂ OC(O)NR--), then the carboxyl (--COOH) groupof a suitably protected amino acid is first reduced to the --CH₂ OHgroup which is then converted by conventional methods to a --OC(O)Clfunctionality or a para-nitrocarbonate --OC(O)O--C₆ H₄ -p-NO₂functionality. Reaction of either of such functional groups with thefree amine or an alkylated amine on the N-terminus of the partiallyfabricated peptide found on the solid support leads to the formation ofa --CH₂ OC(O)NR-- linkage. For a more detailed description of theformation of such --CH₂ -carbamate linkages, see Cho, et al., Science,261:1303-1305 (1993).

Similarly, replacement of an amido linkage in the peptide with aphosphonate linkage can be achieved in the manner set forth in U.S.patent application Ser. Nos. 07/943,805, 08/081,577, and 08/119,700, thedisclosures of which are incorporated herein by reference in theirentirety.

Replacement of an amido linkage in the peptide with a --CH₂ -sulfonamidelinkage can be achieved by reducing the carboxyl (--COOH) group of asuitably protected amino acid to the --CH₂ OH group and the hydroxylgroup is then converted to a suitable leaving group such as a tosylgroup by conventional methods. Reaction of the tosylated derivativewith, for example, thioacetic acid followed by hydrolysis and oxidativechlorination will provide for the --CH₂ --S(O)₂ Cl functional groupwhich replaces the carboxyl group of the otherwise suitably protectedamino acid. Use of this suitably protected amino acid analogue inpeptide synthesis provides for inclusion of an --CH₂ S(O)₂ NR-- linkagewhich replaces the amido linkage in the peptide thereby providing apeptide mimetic. For a more complete description on the conversion ofthe carboxyl group of the amino acid to a --CH₂ S(O)₂ Cl group, see, forexample, Weinstein, B., Chemistry & Biochemistry of Amino Acids,Peptides and Proteins, Vol. 7, pp. 267-357, Marcel Dekker, Inc., NewYork (1983) which is incorporated herein by reference.

Replacement of an amido linkage in the peptide with a urea linkage canbe achieved in the manner set forth in U.S. patent application Ser. No.08/147,805 which application is incorporated herein by reference in itsentirety.

Secondary amine linkages wherein a --CH₂ NH-- linkage replaces the amidolinkage in the peptide can be prepared by employing, for example, asuitably protected dipeptide analogue wherein the carbonyl bond of theamido linkage has been reduced to a CH₂ group by conventional methods.For example, in the case of diglycine, reduction of the amide to theamine will yield after deprotection H₂ NCH₂ CH₂ NHCH₂ COOH which is thenused in N-protected form in the next coupling reaction. The preparationof such analogues by reduction of the carbonyl group of the amidolinkage in the dipeptide is well known in the art (see, e.g., Michael W.Remington, Meth. in Mol. Bio., 35:241-247 (1994)).

The suitably protected amino acid analogue is employed in theconventional peptide synthesis in the same manner as would thecorresponding amino acid. For example, typically about 3 equivalents ofthe protected amino acid analogue are employed in this reaction. Aninert organic diluent such as methylene chloride or DMF is employed and,when an acid is generated as a reaction by-product, the reaction solventwill typically contain an excess amount of a tertiary amine to scavengethe acid generated during the reaction. One particularly preferredtertiary amine is diisopropylethylamine which is typically employed inabout 10 fold excess. The reaction results in incorporation into thepeptide mimetic of an amino acid analogue having a non-peptidyl linkage.Such substitution can be repeated as desired such that from zero to allof the amido bonds in the peptide have been replaced by non-amido bonds.

One can also cyclize the peptides of the invention, or incorporate adesamino or descarboxy residue at the terminii of the peptide, so thatthere is no terminal amino or carboxyl group, to decrease susceptibilityto proteases or to restrict the conformation of the peptide. C-terminalfunctional groups of the compounds of the present invention includeamide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy,and carboxy, and the lower ester derivatives thereof, and thepharmaceutically acceptable salts thereof. Examples of cyclizedcompounds are provided in Tables 4, 5, 6, 8, and 9.

E. DISULFIDE BOND FORMATION

The compounds of the present invention may exist in a cyclized form withan intramolecular disulfide bond between the thiol groups of thecysteines. Alternatively, an intermolecular disulfide bond between thethiol groups of the cysteines can be produced to yield a dimeric (orhigher oligomeric) compound. One or more of the cysteine residues mayalso be substituted with a homocysteine. These intramolecular orintermolecular disulfide derivatives can be represented schematically asshown below: ##STR3## wherein m and n are independently 1 or 2.

Other embodiments of this invention provide for analogs of thesedisulfide derivatives in which one of the sulfurs has been replaced by aCH₂ group or other isostere for sulfur. These analogs can be made via anintramolecular or intermolecular displacement, using methods known inthe art as shown below:

wherein p is 1 or 2. One of skill in the art will readily appreciatethat this displacement can also occur using other homologs of thea-amino-g-butyric acid derivative shown above and homocysteine (see,e.g., Frank A. Robey, Meth. in Mol. Bio., 35(6):73-90 (1990).

Alternatively, the amino-terminus of the peptide can be capped with analpha-substituted acetic acid, wherein the alpha substituent is aleaving group, such as an a-haloacetic acid, for example, a-chloroaceticacid, a-bromoacetic acid, or a-iodoacetic acid. The compounds of thepresent invention can be cyclized or dimerized via displacement of theleaving group by the sulfur of the cysteine or homocysteine residue.See, e.g., Andreu, et al., Meth. in Mol. Bio., 35(7):91-169 (1994);Barker, et al., J. Med. Chem., 35:2040-2048 (1992) and Or, et al., J.Org. Chem., 56:3146-3149 (1991), each of which is incorporated herein byreference. Examples of dimerized compounds are provided in Tables 7, 9,and 10. ##STR4## V. UTILITY

The compounds of the invention are useful in vitro as unique tools forunderstanding the biological role of TPO, including the evaluation ofthe many factors thought to influence, and be influenced by, theproduction of TPO and the receptor binding process. The presentcompounds are also useful in the development of other compounds thatbind to and activate the TPO-R, because the present compounds provideimportant information on the relationship between structure and activitythat should facilitate such development.

The compounds are also useful as competitive binders in assays to screenfor new TPO receptor agonists. In such assay embodiments, the compoundsof the invention can be used without modification or can be modified ina variety of ways; for example, by labeling, such as covalently ornon-covalently joining a moiety which directly or indirectly provides adetectable signal. In any of these assays, the materials thereto can belabeled either directly or indirectly. Possibilities for direct labelinginclude label groups such as: radiolabels such as ¹²⁵ I, enzymes (U.S.Pat. No. 3,645,090) such as peroxidase and alkaline phosphatase, andfluorescent labels (U.S. Pat. No. 3,940,475) capable of monitoring thechange in fluorescence intensity, wavelength shift, or fluorescencepolarization. Possibilities for indirect labeling include biotinylationof one constituent followed by binding to avidin coupled to one of theabove label groups. The compounds may also include spacers or linkers incases where the compounds are to be attached to a solid support.

Moreover, based on their ability to bind to the TPO receptor, thepeptides of the present invention can be used as reagents for detectingTPO receptors on living cells, fixed cells, in biological fluids, intissue homogenates, in purified, natural biological materials, etc. Forexample, by labelling such peptides, one can identify cells having TPO-Ron their surfaces. In addition, based on their ability to bind the TPOreceptor, the peptides of the present invention can be used in in situstaining, FACS (fluorescence-activated cell sorting), Western blotting,ELISA, etc. In addition, based on their ability to bind to the TPOreceptor, the peptides of the present invention can be used in receptorpurification, or in purifying cells expressing TPO receptors on the cellsurface (or inside permeabilized cells).

The compounds of the present invention can also be utilized ascommercial reagents for various medical research and diagnostic uses.Such uses include but are not limited to: (1) use as a calibrationstandard for quantitating the activities of candidate TPO agonists in avariety of functional assays; (2) use to maintain the proliferation andgrowth of TPO-dependent cell lines; (3) use in structural analysis ofthe TPO-receptor through co-crystallization; (4) use to investigate themechanism of TPO signal transduction/receptor activation; and (5) otherresearch and diagnostic applications wherein the TPO-receptor ispreferably activated or such activation is conveniently calibratedagainst a known quantity of a TPO agonist, and the like.

The compounds of the present invention can be used for the in vitroexpansion of megakaryocytes and their committed progenitors, both inconjunction with additional cytokines or on their own. See, e.g.,DiGiusto, et al., PCT Publication No. 95/05843, which is incorporatedherein by reference. Chemotherapy and radiation therapies causethrombocytopenia by killing the rapidly dividing, more mature populationof megakaryocytes. However, these therapeutic treatments can also reducethe number and viability of the immature, less mitotically activemegakaryocyte precursor cells. Thus, amelioration of thethrombocytopenia by TPO or the compounds of the present invention can behastened by infusing patients post chemotherapy or radiation therapywith a population of his or her own cells enriched for megakaryocytesand immature precursors by in vitro culture.

The compounds of the invention can also be administered to warm bloodedanimals, including humans, to activate the TPO-R in vivo. Thus, thepresent invention encompasses methods for therapeutic treatment of TPOrelated disorders that comprise administering a compound of theinvention in amounts sufficient to mimic the effect of TPO on TPO-R invivo. For example, the peptides and compounds of the invention can beadministered to treat a variety of hematological disorders, includingbut not limited to platelet disorders and thrombocytopenia, particularlywhen associated with bone marrow transfusions, radiation therapy, andchemotherapy.

In some embodiments of the invention, TPO antagonists are preferablyfirst administered to patients undergoing chemotherapy or radiationtherapy, followed by administration of the tpo agonists of theinvention.

The activity of the compounds of the present invention can be evaluatedeither in vitro or in vivo in one of the numerous models described inMcDonald, Am. J. of Pediatric Hematology/Oncology, 14:8-21 (1992), whichis incorporated herein by reference.

According to one embodiment, the compositions of the present inventionare useful for treating thrombocytopenia associated with bone marrowtransfusions, radiation therapy, or chemotherapy. The compoundstypically will be administered prophylactically prior to chemotherapy,radiation therapy, or bone marrow transplant or after such exposure.

Accordingly, the present invention also provides pharmaceuticalcompositions comprising, as an active ingredient, at least one of thepeptides or peptide mimetics of the invention in association with apharmaceutical carrier or diluent. The compounds of this invention canbe administered by oral, pulmonary, parental (intramuscular,intraperitoneal, intravenous (IV) or subcutaneous injection), inhalation(via a fine powder formulation), transdermal, nasal, vaginal, rectal, orsublingual routes of administration and can be formulated in dosageforms appropriate for each route of administration. See, e.g.,Bernstein, et al., PCT Patent Publication No. WO 93/25221; Pitt, et al.,PCT Patent Publication No. WO 94/17784; and Pitt, et al., EuropeanPatent Application 613,683, each of which is incorporated herein byreference.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is admixed with at least one inert pharmaceutically acceptablecarrier such as sucrose, lactose, or starch. Such dosage forms can alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., lubricating agents such as magnesium stearate. In thecase of capsules, tablets, and pills, the dosage forms may also comprisebuffering agents. Tablets and pills can additionally be prepared withenteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, with the elixirscontaining inert diluents commonly used in the art, such as water.Besides such inert diluents, compositions can also include adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening, flavoring, and perfuming agents.

Preparations according to this invention for parental administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Suchdosage forms may also contain adjuvants such as preserving, wetting,emulsifying, and dispersing agents. They may be sterilized by, forexample, filtration through a bacteria retaining filter, byincorporating sterilizing agents into the compositions, by irradiatingthe compositions, or by heating the compositions. They can also bemanufactured using sterile water, or some other sterile injectablemedium, immediately before use.

Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to the active substance,excipients such as cocoa butter or a suppository wax. Compositions fornasal or sublingual administration are also prepared with standardexcipients well known in the art.

The compositions containing the compounds can be administered forprophylactic and/or therapeutic treatments. In therapeutic applications,compositions are administered to a patient already suffering from adisease, as described above, in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications. Anamount adequate to accomplish this is defined as "therapeuticallyeffective dose". Amounts effective for this use will depend on theseverity of the disease and the weight and general state of the patient.

The compositions of the invention can also be microencapsulated by, forexample, the method of Tice and Bibi (in Treatise on Controlled DrugDelivery, ed. A. Kydonieus, Marcel Dekker, New York (1992), pp.315-339).

In prophylactic applications, compositions containing the compounds ofthe invention are administered to a patient susceptible to or otherwiseat risk of a particular disease. Such an amount is defined to be a"prophylactically effective dose". In this use, the precise amountsagain depend on the patient's state of health and weight.

The quantities of the TPO agonist necessary for effective therapy willdepend upon many different factors, including means of administration,target site, physiological state of the patient, and other medicantsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in situ administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described, e.g., in Gilman, et al. (eds),Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8thed., Pergamon Press (1990); and Remington's Pharmaceutical Sciences, 7thEd., Mack Publishing Co., Easton, Pa. (1985); each of which is herebyincorporated by reference.

The peptides and peptide mimetics of this invention are effective intreating TPO mediated conditions when administered at a dosage range offrom about 0.001 mg to about 10 mg/kg of body weight per day. Thespecific dose employed is regulated by the particular condition beingtreated, the route of administration as well as by the judgement of theattending clinician depending upon factors such as the severity of thecondition, the age and general condition of the patient, and the like.

Although only preferred embodiments of the invention are specificallydescribed above, it will be appreciated that modifications andvariations of the invention are possible without departing from thespirit and intended scope of the invention.

EXAMPLE 1 Solid Phase Peptide Synthesis

Various peptides of the invention were synthesized using the Merrifieldsolid phase synthesis techniques (see Steward and Young, Solid PhasePeptide Synthesis, 2d. edition, Pierce Chemical, Rockford, Ill. (1984)and Merrifield, J. Am. Chem. Soc., 85:2149 (1963)) or an AppliedBiosystems Inc. Model 431A or 433A peptide synthesizer. The peptideswere assembled using standard protocols of the Applied Biosystems Inc.Synth Assist™ 1.0.0 or Synth Assist™ 2.0.2. Each coupling was performedfor 2×30 min. with HBTU(2-(1H-benzatriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate) and HOBt (1-hydroxybenzotriazole).

The resin used was HMP resin (p-hydroxymethyl phenoxymethyl)polystyreneresin or PAL (Milligen/Biosearch), which is a cross-linked polystyreneresin with 5-(4'-Fmoc-aminomethyl-3,5'-dimethyoxyphenoxy)valeric acid asa linker. Use of PAL resin results in a carboxyl terminal amidefunctionality upon cleavage of the peptide from the resin. Uponcleavage, the HMP resin produces a carboxylic acid moiety at theC-terminus of the final product. Most reagents, resins, and protectedamino acids (free or on the resin) were purchased from Millipore orApplied Biosystems Inc.

The Fmoc group was used for amino protection during the couplingprocedure. Primary amine protection on amino acids was achieved withFmoc and side chain protection groups were t-butyl for serine, tyrosine,glutamic acid, and threonine; trityl for glutamine; Pmc(2,2,5,7,8-pentamethylchroman-6-sulfonyl) for arginine;N-t-butyloxycarbonyl for tryptophan; N-trityl for histidine and S-tritylfor cysteine.

Removal of the peptides from the resin and simultaneous deprotection ofthe side chain functions were achieved by treatment with reagent K orslight modifications of it. Alternatively, in the synthesis of thosepeptides, with an amidated carboxyl terminus, the fully assembledpeptide was cleaved with a mixture of 90% trifluoroacetic acid, 5%ethanedithiol, and 5% water, initially at 4° C., and graduallyincreasing to room temperature. The deprotected peptides wereprecipitated with diethyl ether. In all cases, purification was bypreparative, reverse-phase, high performance liquid chromatography on aC₁₈ bonded silica gel column with a gradient of acetonitrile/water in0.1% trifluoroacetic acid. The homogeneous peptides were characterizedby Fast Atom Bombardment mass spectrometry or electrospray massspectrometry and amino acid analysis when applicable.

In a preferred embodiment, the peptides of this invention are dimerizedusing standard synthetic procedures known to and used by those of skillin the art. Exemplar synthetic schemes for preparing the dimer peptidecompounds of this invention are set forth in FIGS. 26 and 27. Followingthese synthetic schemes, those of skill in the art can readily preparedimer peptide compounds in accordance with the present invention. Inaddition, it will be readily apparent to those of skill in the art thatthe dimeric subunits can readily be linked using methodologies andlinkers other than those described in FIGS. 26 and 27.

Pegylation of the Peptides

A. Preparation of di-PEGylated AF13948 with PEG2 branched PEG, 20,000 Mw(see, FIG. 15)

AF13948 was dissolved in 100 mM bicine pH 8.0 at a concentration of 10mg/ml, added to a 1.25 fold molar excess of powdered PEG2 (commerciallyavailable from Shearwater Polymers, Inc. (Huntsville, Ala.)) and stirredat room temperature until the reaction was complete, typically 1-2hours. The reaction was monitored by reverse phase HPLC using a 40-65%acetonitrile gradient with a YMC ODS AQ column. When the reaction wascomplete, the solution was added to a second 1.25 molar excess ofpowdered PEG2 and the process was repeated 4 times using a total of 5moles of PEG2 for each mole of AF13948. The solution was diluted 2 foldwith PBS to reduce the viscosity and loaded onto a superdex 200 column(Pharmacia), previously equilibrated and eluted with PBS. Fractions fromthe size exclusion column were analyzed by reverse phase HPLC. Fractionscontaining di-PEG-AF13948 which eluted prior to any mono-PEG-AF13948were pooled and stored at 5° C. or lyophilized.

B. Preparation of di-PEGylated AF13948 with SPA-mPEG 20,000 Mw (see,FIG. 16)

AF13948 was dissolved in 100 mM bicine pH 8.0 at a concentration of 10mg/ml, added to a 5 fold molar excess of powdered SPA-mPEG (commerciallyavailable from Shearwater Polymers, Inc. (Huntsville, Ala.)) and stirredat room temperature until the reaction was complete, typically 2 hours.The reaction was monitored by reverse phase (YMC ODS AQ) HPLC and whenit was complete, the solution was diluted 10 fold with deionized waterand loaded onto a SP-sepharose column equilibrated in 2 mM sodiumphosphate buffer at pH 7.0. The unreactded PEG and the NHS flowedthrough the column. The PEGylated AF13948 was then eluted using a 0 to150 mM NaCl gradient, with the di-PEGylated form eluting before anymono-PEGylated material. The fractions were analyzed by reverse phaseHPLC and the appropriate fractions combined. The di-PEG-AF13948 wasbuffer exchanged into PBS and either stored at 5° C. or lyophilized.

C. Preparation of di-PEGylated AF13848 with mPEG aldehyde 20,000 Mw(see, FIG. 17)

AF13948 was dissolved in 100 mM sodium phosphate pH 7.0 at aconcentration of 10 mg/ml, added to a 6 fold molar excess of powderedPEG aldehyde (commercially available from Shearwater Polymers, Inc.(Huntsville, Ala.)) and 1M sodium cyanoborohydride solution was added togive a final sodium cyanoborohydride concentration of 100 mM. Thesolution was stirred for 18 hours at room temperature, then bufferexchanged into PBS, using a 3K (Amicon YM) ultrafiltration membrane. Thesolution ws diluted 2 fold with PBS to reduce the viscosity and loadedon a superdex 200 column (Pharmacia), previously equilibrated and elutedwith PBS. Fractions from the size exclusion column were analyzed byreverse phase HPLC. Fractions containing di-PEG-AF13948 which elutedprior to any mono-PEG-AF13948 were pooled and stored at 5° C. orlyophilized.

EXAMPLE 2 Bioassays

Bioactivity of the peptides can be measured using a thrombopoietindependent cell proliferation assay. Murine IL-3 dependent Ba/F3 cellswere transfected with full length human TPO-R. In the absence of IL-3(WEHI-3 conditioned media), these cells are dependent on TPO forproliferation. The parental, untransfected cell line does not respond tohuman TPO, but remains IL-3 dependent.

Bioassays have been performed on both of the above cell lines usingsynthetic peptides derived from library screening. The cells were grownin complete RPMI-10 media, containing 10% WEHI-3 conditioned media, thenwashed twice in PBS, resuspended in media which lacked WEHI-3conditioned media, and added to wells containing dilutions of peptide orTPO at 2×10⁴ cells/well. The cells were incubated for 48 hours at 37° C.in a humidified 5% CO₂ atmosphere and metabolic activity was assayed bythe reduction of MTT to formazan, with absorbance at 570 Nm measured onan ELISA plate reader. The peptides tested stimulated proliferation ofTPO-R transfected Ba/F3 cells in a dose dependent manner as shown inFIG. 1. These peptides have no effect on the parental cell line.

EXAMPLE 3 Binding Affinity

Binding affinities of chemically synthesized peptides for TPO-R weremeasured in a competition binding assay. The wells of a microtiter platewere coated with 1 mg streptavidin, blocked with PBS/1% BSA, followed by50 ng of biotinylated anti-receptor immobilizing antibody (Ab179). Thewells were then treated with a 1:10 dilution of soluble TPO-R harvest.Various concentrations of peptide or peptide mimetic were mixed with aconstant amount of a truncated form of TPO consisting of residues 1-156fused to the C-terminus of maltose binding protein (MBP-TPO₁₅₆). Thepeptide MBP-TPO₁₅₆ mixtures were added to the TPO-R coated wells,incubated for 2 hours at 4° C. and then washed with PBS. The amount ofMBP-TPO₁₅₆ that was bound at equilibrium was measured by adding a rabbitanti-sera directed against MBP, followed by alkaline phosphataseconjugated goat anti-rabbit IgG. The amount of alkaline phosphatase ineach well was then determined using standard methods.

The assay is conducted over a range of peptide concentrations and theresults are graphed such that the y axis represents the amount of boundMBP-TPO₁₅₆ and the x axis represents the concentration of peptide orpeptide mimetic. One can then determine the concentration at which thepeptide or peptide mimetic will reduce by 50% (IC₅₀) the amount ofMBP-TPO₁₅₆ bound to immobilized TPO-R. The dissociation constant (Kd)for the peptide should be similar to the measured IC₅₀ using the assayconditions described above.

EXAMPLE 4 "Peptides on Plasmids"

The pJS142 vector is used for library construction and is shown in FIG.4. Three oligonucleotide sequences (SEQ ID NOS 169-171, respectively)are needed for library construction: ON-829 (5' ACC ACC TCC GG); ON-830(5' TTA CTT AGT TA) and a library specific oligonucleotide of interest(5' GA GGT GGT {NNK}_(n) TAA CTA AGT AAA GC), where {NNK}_(n) denotes arandom region of the desired length and sequence. The oligonucleotidescan be 5' phosphorylated chemically during synthesis or afterpurification with polynucleotide kinase. They are then annealed at a1:1:1 molar ratio and ligated to the vector.

The strain of E. coli which is preferably used for panning has thegenotype: Δ(srl-recA) endAl nupG lon-11 sulA1 hsdR17 Δ(ompT-fepC)266ΔclpA319::kan ΔlacI lac ZU118 which can be prepared from an E. colistrain from the E. coli Genetic Stock Center at Yale University (E. colib/r, stock center designation CGSC:6573) with genotype lon-11 sulA1. Theabove E. coli strain is prepared for use in electroporation as describedby Dower, et al., Nucleic Acids Res., 16:6127 (1988), except that 10%glycerol is used for all wash steps. The cells are tested for efficiencyusing 1 pg of a Bluescript plasmid (Stratagene). These cells are usedfor growth of the original library and for amplification of the enrichedpopulation after each round of panning.

Peptides on plasmids are released from cells for panning by gentleenzymatic digestion of the cell wall using lysozyme. After pelleting ofthe cell debris, the crude lysate can be used directly on mostreceptors. If some additional purification of the plasmid complexes isneeded, a gel filtration column can be used to remove many of the lowmolecular weight contaminants in the crude lysate.

Panning is carried out in a buffer (HEKL) of a lower salt concentrationthan most physiological buffers. The panning can be conducted inmicrotiter wells with a receptor immobilized on a nonblocking monoclonalantibody (MAb) or by panning on beads or on columns. More specifically,in the first round of panning, 24 wells, each coated with receptor, canbe used. For the second round, six wells coated with receptor (PANsample) and 6 wells without receptor (NC sample) are typically used.Comparison of the number of plasmids in these two samples can give anindication of whether receptor specific clones are being enriched bypanning. "Enrichment" is defined as the ratio of PAN transformants tothose recovered from the NC sample. Enrichment of 10 fold is usually anindication that receptor specific clones are present.

In later rounds of panning, it is useful to reduce the input of lysateinto the wells to lower nonspecific background binding of the plasmidcomplexes. In round 2, usually 100 μl of lysate per well is used. Inround 3, 100 μl of lysate per well diluted with 1/10 in HEKL/BSA isused. For further rounds of panning, typically an input of plasmidtransforming units of at least 1000 fold above the estimated remainingdiversity is used.

The binding properties of the peptides encoded by individual clones aretypically examined after 3, 4, or 5 rounds of panning, depending on theenrichment numbers observed. Typically, an ELISA that detects receptorspecific binding by LacI-peptide fusion proteins is used. LacI isnormally a tetramer and the minimum functional DNA binding species is adimer. The peptides are thus displayed multivalently on the fusionprotein. Assuming that a sufficient density of receptor can beimmobilized in wells, the peptides fused to LacI will bind to thesurface in a cooperative, multivalent fashion. This cooperative bindingpermits the detection of binding events of low intrinsic affinity. Thesensitivity of this assay is an advantage in that initial hits of lowaffinity can be easily identified, but is a disadvantage in that thesignal in the ELISA is not correlated with the intrinsic affinity of thepeptides. Fusion of the peptides to maltose binding protein (MBP) asdescribed below permits testing in an ELISA format where signal strengthis better correlated with affinity. See FIGS. 5A-B.

DNA from clones of interest can be prepared in double stranded formusing any standard miniprep procedure. The coding sequences ofinteresting single clones or populations of clones can be transferred tovectors that fuse those sequences in frame with the gene encoding MBP, aprotein that generally occurs as a monomer in solution. The cloning of alibrary into PJS142 creates a BspEI restriction site near the beginningof the random coding region of the library. Digestion with BspEI andScaI allows the purification of a .sup.˜ 900 bp DNA fragment that can besubcloned into one of two vectors, pELM3 (cytoplasmic) or pELM15(periplasmic), which are simple modifications of the pMALc2 and pMALp2vectors, respectively, available commercially from New England Biolabs.See FIGS. 5A-B. Digestion of pELM3 and pELM15 with AgeI and ScaI allowsefficient cloning of the BspEI-ScaI fragment from the pJS142 library.The BspEI and AgeI ends are compatible for ligation. In addition,correct ligation of the ScaI sites is essential to recreate a functionalbla (Amp resistance) gene, thus lowering the level of background clonesfrom undesired ligation events. Expression of the tac promoter-drivenMBP-peptide fusions can then be induced with IPTG.

Lysates for the LacI or MBP ELISAs are prepared from individual clonesby lysing cells using lysozyme and removing insoluble cell debris bycentrifugation. The lysates are then added to wells containingimmobilized receptor and to control wells without receptor. Binding bythe LacI or MBP peptide fusions is detected by incubation with a rabbitpolyclonal antiserum directed against either LacI or MBP followed byincubation with alkaline phosphatase labeled goat anti rabbit secondantibody. The bound alkaline phosphatase is detected with p-nitrophenylphosphate chromogenic substrate.

EXAMPLE 5 "Headpiece Dimer" System

A variant of the LacI peptides-on-plasmids technique utilizes a DNAbinding protein called "headpiece dimer". DNA binding by the E. coli lacrepressor is mediated by the approximately 60 amino acid "headpiece"domain. The dimer of the headpiece domains that binds to the lacoperator is normally formed by association of the much largerapproximately 300 amino acid C-terminal domain. The "headpiece dimer"system utilizes headpiece dimer molecules containing two headpiecesconnected via short peptide linker. These proteins bind DNA withsufficient stability to allow association of a peptide epitope displayedat the C-terminus of the headpiece dimer with the plasmid encoding thatpeptide.

The random peptides are fused to the C-terminus of the headpiece dimer,which binds to the plasmid that encoded it to make a peptide-headpiecedimer-plasmid complex that can be screened by panning. The headpiecedimer peptides-on-plasmids system allows greater selectivity for highaffinity ligands than the LacI system. Thus, the headpiece dimer systemis useful for making mutagenesis libraries based on initial low-affinityhits, and selecting higher affinity variants of those initial sequences.

The libraries are constructed as with peptides on plasmids usingheadpiece diner vector pCMG14 (see FIGS. 6A-C). The presence of the lacoperator is not required for plasmid binding by the headpiece dimerprotein. The libraries were introduced into bacterial strain comprisingE. coli (lon-11 sulA1 hsdR17 (ompT-fepC) ΔclpA319::kan ΔlacI lac ZU118Δ(srl-recA) 306::Tn10 and amplified under conditions of basal (A)promoter induction. Panning of headpiece dimer libraries is carried outby similar procedures to those used for LacI libraries, except that HEKbuffer is used instead of HEKL buffer and elution of plasmids from thewells is performed with aqueous phenol instead of with IPTG. Sequencesfrom headpiece dimer panning are often characterized after transfer tothe MBP vector so that they can be tested in the affinity sensitive MBPELISA and also so that populations of clones can be screened by colonylifts with labeled receptor.

EXAMPLE 6

In this example cyclized compounds were subjected to three assays.First, IC₅₀ valves were obtained as described above. Additionally, anMTT cell proliferation assay as described above was performed tocalculate EC₅₀ values. Finally, a microphysiometer (Molecular DevicesCorp.) assay was performed. Basically, in this assay the rate ofacidification of the extracellular medium in response to TPO receptorstimulation by the compounds of the invention was determined. The rangesfor EC₅₀ are symbolically indicated as for IC₅₀ described above. Theresults are summarized in Table 4.

                                      TABLE 4    __________________________________________________________________________                                  EC50 (nM)                                        EC50 (nM)    Structure                     Proliferation                                        Microphys.                                              IC50 (nM)    __________________________________________________________________________     ##STR5##                     ++    ++    ++     ##STR6##                     ++    ++    ++     ##STR7##                     ++    ++    ND     ##STR8##                     +     +     ##STR9##                     +     +     ND     ##STR10##                    +     +     ++     ##STR11##                    +     +     ++     ##STR12##                    +     +     ++     ##STR13##                    +     +     ++     ##STR14##                    +     +     +     ##STR15##                    ++    +     ++     ##STR16##                    +     +     ND     ##STR17##                    +     +     ND     ##STR18##                    ++    +     ND     ##STR19##                    ++    +     ND     ##STR20##                    +     +     +     ##STR21##                    +     +     ND    __________________________________________________________________________

EXAMPLE 7

In this example amino acid substitutes at positions D, E, I, S, or F inthe cyclized compound (SEQ ID NO:12) ##STR22## were assayed for EC₅₀ andIC₅₀ values as described above. Microphysiometer results are given inparentheses. The results are summarized in Table 5 below.

                  TABLE 5    ______________________________________     ##STR23##                      EC50 (nM)    Substitution      Cell Prolif.                                IC50 (nM)    ______________________________________    E - Q             ++(+)     ++    D - A             +(+)      ++    I - A             +(+)      +    S - A             ++(++)    ++    S - DAla          +         +    S - Sar           +         ++    S - Aib           ++(+)     ++    S - DSer          ++        ++    S - Nva           ++(++)    ++    S - Abu           ++        ++    S - (NMeAla)      +         +    S - (NMeVal)      +         +    S - (NMeAla)*     +         +    S - (NorLeu)      ++        ++    S - (t-BuGly)     +         ++    S -  NMeSer(Bzl)!           +    S - (Homoser)     ND        ND    S - (NMeLeu)      +         ND    F - A             +(+)      ++    F - DAla          +         ++    F - DPhe          +         ++    F - Homo - Phe    ++(++)    ++    F - CHA           ++(++)    ++    F - Thi           ++        ++    F - (Ser(Bzl))    ++        ++    F - (NMeAla)      +         +    F - (Phenylgly)   ++(++)    ++    F - (Pyridylala)  ++        ++    F - (p-Nitrophe)  ++(++)    ++    F - (3,4-di-ClPhe)                      ++(+)     ++    F - (p-ClPhe)     ++        ++    F - (2-Nal)       ++(++)    ++    F - (1-Nal)       ++        ++    F - (DiPhAla)     ++        ++    F - (NMePhe)      ++        ND    S,F - Ava (thioether)                      +         ++    S,F - Ava(cyscys) +         ++    S,F - Ava         +         ++    AD - deletion     +(+)      ND    ADG - deletion    (+)       +    ______________________________________     ##STR24##

EXAMPLE 8

In this example, amino acid substitutions in the compound (SEQ ID NO:173) ##STR25## were evaluated at positions D, S, or F as indicated inTable 6 below. EC₅₀ and IC₅₀ values were calculated as described above.Microphysiometer results are in parentheses.

                  TABLE 6    ______________________________________     ##STR26##                      EC50 (nM)    Substitution      Cell Prolif.                                IC50 (nM)    ______________________________________    D - E             (+)       ND    free acid form    ++(+)     ND    C-term. Gly addition                      ++        ++    S - Abu           ++(++)    ND    F - DiPhAla       (++)      ++    S,F - Abu,DiPhAla +(+)      ++    ______________________________________

EXAMPLE 9

In this example EC₅₀ and IC₅₀ values were calculated as described abovefor the dimer compounds listed in Table 7 below. The cyclized monomer(SEQ ID NO: 173) ##STR27## is included as a comparison.

In Table 8, EC₅₀ and IC₅₀ values determined as described above forcyclized and dimerized variants of I E G P T L R Q W L A A R A arecompared.

In Table 9, truncations of the dimer ##STR28## are compared. EC₅₀ andIC₅₀ values were calculated as described above. Microphysiometer resultsare given in parentheses.

It should be noted that in the dimerized peptides compounds set forthin, for example, Tables 9, 13 and 14, each of the dimeric subunits isattached to a lysine linker, with one of the dimeric subunits beingattached to the α-amino group of the lysine and the other dimericsubunit being attached to the ε-amino group of the lysine.

                                      TABLE 7    __________________________________________________________________________                                  EC50 (nM)                                  Microphys.                                        Prolif.                                            IC50 (nM)    __________________________________________________________________________     ##STR29##                    ++    ++  ++     ##STR30##                    ++    ++  ++     ##STR31##                    ++    ++  ++     ##STR32##                    ++    ++  ++     ##STR33##                    ++    ++  ++     ##STR34##                    ND    +   +     ##STR35##                    ++    ++  ++     ##STR36##                    ND    ++  ++     ##STR37##                    ++    ++  ++     ##STR38##                    ++    ++  ++     ##STR39##                    ND    ++  ++     ##STR40##                    ++    ++  +     ##STR41##                    ND    ++  ++     ##STR42##                    ++    ++  +     ##STR43##                    +     +   +     ##STR44##                    ++    +   +    __________________________________________________________________________

                                      TABLE 8    __________________________________________________________________________                                  EC50 (nM)                                  Microphys.                                        Prolif.                                            IC50 (nM)    __________________________________________________________________________    (H)IEGPTLRQWLAARA(NH.sub.2)(SEQ ID NO:193)                                  N.D.  ++  ++     ##STR45##                    N.D.  ++  ++     ##STR46##                    ++    ++  ++     ##STR47##                    ++    ++  ++    __________________________________________________________________________

                                      TABLE 9    __________________________________________________________________________     ##STR48##                                  EC50 (nM)    Sequence                      Cell Prolif.                                        IC50 (nM)    __________________________________________________________________________     ##STR49##                    ++    ND     ##STR50##                    ++    ND     ##STR51##                    ++(++)                                        ND     ##STR52##                    ND    ND     ##STR53##                    ++    ND     ##STR54##                    ++(++)                                        ND     ##STR55##                    ++    ND     ##STR56##                    ++    ND    __________________________________________________________________________

EXAMPLE 10

In this example various substitutions were introduced at positions G, P,and W in the cyclized compound ##STR57##

Table 10 lists examples of the substituted compounds that show TPOagonist activity. The substitutions abbreviated in the table are asfollows:

                  TABLE 10    ______________________________________     ##STR58##    G               P            W    ______________________________________    Sar             Hyp(OBn)     Nal    Sar             Hyp(OBn)     Nal    Gly             Pro          Trp    Gly             Pro          Trp    Sar             Hyp(OBn)     Nal    Gaba            Pro          Trp    CprGly          Pro          Trp    Sar             Hyp(OBn)     Nal    Gly             Pro          Trp    Gly             Pro          Nal    Sar             Pro          Trp    CprGly          L-Tic        Nal    Gly             D-Tic        D-Trp    CprGly          D-Tic        Trp    Gaba            Hyp(OBn)     Trp    ______________________________________     ##STR59##

EXAMPLE 11

To assess the feasibility of mice as a convenient test species, severalin vitro experiments, designed to measure the activity of the testcompounds on the mouse receptor, have been done. First, marrow cells,harvested from the femurs of 8 to 9 week one Balb/C mice, were incubatedfor 7 days in liquid culture with either rhuTPO or variousconcentrations of the test peptides. At the end of the incubationperiod, the cultures were concentrated by Cytospin, stained foracetylcholinesterase (AChE, a diagnostic of mouse megakaryocytes), andcounted by microscopic analysis. One (1) Nm rhuTPO gave rise to theoutgrowth of very large (>40 um) non-adherent cells that stain for AChE.These cells appear to be mature megakaryocytes. From an initial seedingof 10⁶ total marrow cells/ml (in 50 ml cultures) an estimated 1 to 2×10⁶megakaryocytes developed. This response to TPO was designated as"maximal". Control cultures containing no added growth factors producedvery few AChE-positive cells. Several of the peptide compounds weretested at high concentration in this assay and the results aresummarized in Table 11. Peptide A at 10 uM produced a maximal responseof the mouse marrow. This finding was the first evidence that thispeptide family is active on the murine receptor. In a second experiment,marrow cells were harvested and cultured in semi-solid medium(methylcellulose) containing either no factors, 1 nM rhuTPO, or 10 uMPeptide A. After 7 days in culture, colonies of large cell (presumed tobe megakaryocytes) were counted and grouped into small colonies (3-5cells) or large colonies (greater than 6 cells). The results are shownin Table 12. TPO and the test peptides both produced substantially morecolonies of both size than did the negative control cultures. Thisindicates that the peptides mimic TPO in their ability to stimulate theexpansion of the Mk precursor cell population.

To obtain a more quantitative comparison of the activity of the testcompounds on murine and human receptors, the muTPO receptor was clonedand transfected into BaF3 cells. A TPO dependent population of cells wasisolated.

                  TABLE 11    ______________________________________    Peptide    Concentration Tested (nM) Response    ______________________________________    D          100,000        none    C           40,000        maximal**    C + S.A.*    1000         maximal**    S.A. alone   1000         none    B          100,000        minimal    A           10,000        maximal**    TPO (R & D)                   1          "maximal"    ______________________________________     *Streptavidin complexed to biotinylated peptide  concentration of putativ     1:4 complex.     **Compared to recombinant human TPO     **25-30% ACE staining cells on cytospin     No factor cultures  ca. 5% AChE staining cells (lower cellularity)

                  TABLE 12    ______________________________________    Compound       3-5 large cells                              6-12 large cells    ______________________________________    No factors  1       2         1    No factors  2       1         1    1 nM TPO    #1-1   15         6    1 nM TPO    #1-2   12         1    1 nM TPO    #2-1   16         8    1 nM TPO    #2-2   13         3    10 uM Peptide                #1-1   25         10    10 uM Peptide                #1-2   22         8    10 uM Peptide                #2-1   22         7    10 uM Peptide                #2-2   21         10    ______________________________________

EXAMPLE 12

In this example, various substitutions, deletions and additions to thedimer ##STR60## were analyzed using three assays. First, an MTT cellproliferation assay was performed as described above. Second, amicrophysiometer assay was performed as described above. Finally, areporter assay was performed. BaF3/TPOR cells transfected with ac-fos/luciferase reporter plasmid were starved overnight in completeRPMI-10 media containing 0.1% WEHI-3 conditioned media, then washed twotimes in PBS. Cells were resuspended in media which lacked WEHI-3conditioned media, and added to wells containing serial dilutions ofpeptide at 5×10⁵ cells/well. The cells were incubated for two hours at37° in a humidified 5% Co₂ atmosphere, and luciferase expression wasmeasured with a luminometer after addition of the luciferin substrate.

                                      TABLE 13    __________________________________________________________________________                                       EC50                                       (pM)                                           EC50                                       Cell                                           (pM) EC50 (pM)    Peptide Sequence                   Prolif.                                           Reporter                                                Microphys    __________________________________________________________________________     ##STR61##                         ++  ++   ++     ##STR62##                         ++  ++   ++     ##STR63##                         ++  ++   ++     ##STR64##                         ++  ++   ++     ##STR65##                         ++  ++   ND     ##STR66##                         ++  ++   ND     ##STR67##                         ++  ++   ND     ##STR68##                         ++  ++   ++     ##STR69##                         ++  ++   ND     ##STR70##                         ++  ++   ++     ##STR71##                         ++  ++   ND     ##STR72##                         ++  ++   ++     ##STR73##                         ND  ++   ND     ##STR74##                         ND  ++   ++     ##STR75##                         ++  ++   ++     ##STR76##                         ND  ++   ND     ##STR77##                         ++  ++   ++     ##STR78##                         ND  ++   ND     ##STR79##                         ND  ++   ND     ##STR80##                         ND  ++   ND     ##STR81##                         ++  ++   ND     ##STR82##                         ++  ND   ND    __________________________________________________________________________

                                      TABLE 14    __________________________________________________________________________                                       EC50                                       (nM)                                       Cell                                           IC50    Peptide Sequence                   Prolif.                                           (nM)    __________________________________________________________________________     ##STR83##                         ++  ++     ##STR84##                         ++  ++     ##STR85##                         ++  ++     ##STR86##                         ++  ++     ##STR87##                         ++  ++     ##STR88##                         ++  ND     ##STR89##                         ++  ND    __________________________________________________________________________

EXAMPLE 13

In this example, the pharmacokinetic behavior of various PEGylatedpeptides was determined. Pharmacokinetic behavior of compounds is animportant determinant of their pharmacological activity. A key componentof the pharmacokinetic profile is the persistence of the compound in theplasma of laboratory animals. This persistence is usually expressed interms of compound concentration in plasma as a function of time afteradministration.

Throughout these experiments, male 20-25 g Balb/c mice were used. Avolume of 200 μl was injected IV or SC. The vehicle was Dulbecco's PBS;5% DMSO; 0.1% w/v BSA. Plasma was harvested at sacrifice using heparinas anticoagulant.

Compound concentrations were measured using a reporter cell assay.Dilutions of plasma were added to the Baf/3 TPOr/fos/lux construct 3.Luciferase expression was measured with a luminometer after addition ofthe luciferin substrate. The stimulatory activity of the individualplasma samples was converted to a concentration expressed as peptideequivalents of compound per volume of plasma (nM or ng/ml). Thisconcentration was established by comparison with a standard curveconstructed in the reporter assay with the parent compound.

Table 15 and FIG. 10 show the concentrations in plasma of compoundsAF13948 and the di-pegylated AF13948 (polyethylene glycol (PEG) averageMW=5000 D) as a function of time after injection of 700 μg peptide/kg.Administration of AF13948 results in activity in plasma detectable abovethe level present in vehicle-injected mice until 60 min PI. The additionof the 5K PEG increases the concentration in plasma greater than100-fold and extends the time it can be detected in plasma until atleast 240 min PI.

                  TABLE 15    ______________________________________    Plasma concentrations of AF13948, 5K DiPEG-AF13948    following IV injection of 700 μg/kg    expressed as peptide equivalents (ng/ml)    time (min)             DiPEG-AF13948  AF13948  vehicle    ______________________________________    10       361.36         0.80     0.02    30       91.49          0.26     0.02    60       54.59          0.11     0.02    120      16.54          0.04     0.03    240      11.20          0.02     0.01    ______________________________________

Table 16 and FIG. 11 show the concentrations in plasma of compounds 5KDiPEG AF13948 (PEG average MW=5000 D) and the 20K DiPEG AF13948 (PEGaverage MW=20,000 D) as a function of time after injection of 500 μgpeptide/kg. Concentration and persistence in plasma of the 20KPEG-modified compound is greatly increased above that of the 5K DiPEGAF13948.

                  TABLE 16    ______________________________________    Plasma concentrations of AF13948, 5K DiPEG-AF13948    following IV injection of 500 μg/kg    expressed as peptide equivalents (ng/ml)    time (hours)               5K          20K     vehicle    ______________________________________    0.5        54.56       1257.50 0.04    1.5        16.89       3481.31 0.03    4          2.76        1970.46 0.02    8          1.02        1158.59 0.02    16         0.17        259.41  0.03    24         0.13        710.75  0.04    ______________________________________

Table 17 and FIG. 12 show the plasma concentrations of compound 20KDiPEG AF13948 after injection of 100, 10 and 1 μg peptide/kg and extendsthe time of observation to 120 hours PI. Concentrations observed inplasma were found to be proportional to administered doses.Administration of a single IV dose of 100 μg/kg of the compound resultedin elevated plasma concentrations for at least 96 hours PI.

                  TABLE 17    ______________________________________    Plasma concentrations of 20K DiPEG-AF13948 peptide    following IV injection of 1, 10, or 100 μg/kg    expressed as nM DiPEG-13948 peptide equivalents    time (hours)               dose (μg/kg)    ______________________________________    0.08       291.66        32.89  2.93    0.25       550.82        45.48  2.63    0.5        1318.53       29.16  2.33    1.5        328.30        39.12  0.98    3          285.43        25.77  1.39    8          134.60        8.33   0.90    24         106.36        11.79  0.20    48         56.64         0.70   0.06    72         15.19         0.57   0.01    96         0.42          0.01   0.00    120        0.02          0.01   0.00    ______________________________________

Table 18 and FIG. 13 show the plasma concentrations following the SC andIV injection of 10 μg/kg 20K DiPEG AF13948. SC injection of a single 10μg/kg dose resulted in elevated plasma activities for 96 hours PI.Profiles of plasma concentrations achieved with these 2 routes ofadministration indicate the good bioavailability of SC administered 20KDiPEG AF13948.

                  TABLE 18    ______________________________________    Plasma concentrations of 20K DiPEG-AF13948    peptide following IV and SC injection of 10 μg/kg    expressed as nM DiPEG-13948 peptide equivalents    time (hours)      SC     IV    ______________________________________    0.08              0.02   32.89    0.25              0.06   45.48    0.5               0.09   29.16    1.5               0.36   39.12    3                 0.82   25.77    8                 1.48   8.33    24                7.27   11.79    48                1.09   0.70    72                0.03   0.57    96                0.01   0.01    120               0.01   0.01    ______________________________________

In addition, FIG. 14 indicates that GW350781, 5K-PEGylated peptide, hasincreased stability, i.e., an increased half-life, over thenon-PEGylated form of the peptide. As such, FIGS. 10-14 indicate thatthe PEGylated peptides have good bioavailability and increased stabilityin human serum.

EXAMPLE 14

Using the assay described above, the pharmacokinetic profile of apeptide variously derivatized with PEG was determined. In thisexperiment, the peptide was derivatized with branched PEG (di(2)), withan ester linked PEG (SPA) and with an aldehyde linked PEG (ALDH) asillustrated in FIGS. 15-17. FIG. 18 shows the plasma concentrationsfollowing SC injection of 10 μg/kg. From FIG. 18, it is apparent thatall three of the peptide compounds variously derivatized PEG havefavorable pharmacokinetic profiles.

EXAMPLE 15

In this experiment, PEGylated peptides were evaluated for their effecton thrombocytopenia in a mouse model. In this assay, mice are madethrombocytopenic by treating the Balb/C mice with carboplatin (90 mg/kgintraperitoneally) on Day 0. GW35071 (1 mg/kg/day), the 5K-PEGylatedpeptide, and GW305805 (32.5 μg/kg/day), the 20K-diPEGylated peptide,were given on Days 1-9 (s.c., qd). From FIGS. 19-20, it is apparent thatthe PEGylated peptides ameliorate carboplatin-induced thrombocytopeniaon about Day 10. These results clearly indicate that the PEGylatedpeptides of the invention can ameliorate thrombocytopenia in a mousemodel.

EXAMPLE 16

In this experiment, PEGylated peptides were evaluated for their effecton platelet levels in normal mice. In one experiment, GW350781 (1mg/kg/day), the 5K-PEGylated peptide, and GW305805 (32.5 μg/kg/day), the20K-diPEGylated peptide, were given on Days 1-9 (s.c., qd). On days1-15, blood was sampled at intervals by tail vein bleeds. From FIGS.21-22, it is apparent that the PEGylated peptides have an effect onthrombocytosis, with the 20K-diPEGylated peptide being about 100-foldmore potent than the 5K-PEGylated peptide.

In another experiment, GW350781, the 5K-PEGylated peptide, and GW305805,the 20K-diPEGylated peptide, were given on Days 1-5 (s.c., qd). On day6, the animals were sacrificed and peripheral blood platelet counts wereobtained. FIGS. 23-25 set forth the effects of varying doses of thePEGylated peptides as well as the effects of single-dose versusmultiple-dose. Such results clearly indicate that the PEGylated peptidesof the invention can be used to increase platelets in a mouse model.

The disclosures in this application of all articles and references,including patent documents, are incorporated herein by reference intheir entirety for all purposes.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 244    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    TrpSerXaaTrpSer    15    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    XaaXaaXaaXaaXaaXaaXaa    15    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 9 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    XaaGlyXaaXaaXaaXaaXaaTrpXaa    15    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    ProThrLeuArgXaaXaa    15    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    XaaXaaGlyXaaXaaXaaXaaXaaTrpXaa    1510    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    GlyGlyCysAlaAspGlyProThrLeuArgGluTrpIleSerPheCys    151015    GlyGly    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    GlyAsnAlaAspGlyProThrLeuArgGlnTrpLeuGluGlyArgArg    151015    ProLysAsn    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    GlyGlyCysAlaAspGlyProThrLeuArgGluTrpIleSerPheCys    151015    GlyGlyLys    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    ThrIleLysGlyProThrLeuArgGlnTrpLeuLysSerArgGluHis    151015    ThrSer    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    SerIleGluGlyProThrLeuArgGluTrpLeuThrSerArgThrPro    151015    HisSer    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    LeuAlaIleGluGlyProThrLeuArgGlnTrpLeuHisGlyAsnGly    151015    ArgAspThr    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    CysAlaAspGlyProThrLeuArgGluTrpIleSerPheCys    1510    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    IleGluGlyProThrLeuArgGlnTrpLeuAlaAlaArgAla    1510    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    CysXaaXaaXaaXaaXaaXaa    15    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    GlyGlyCysThrLeuArgGluTrpLeuHisGlyGlyPheCysGlyGly    151015    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 8 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    XaaCysXaaXaaXaaXaaXaaXaa    15    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    IleGluGlyProThrLeuArgGlnTrpLeuAlaAlaArgAlaLys    151015    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 15    (D) OTHER INFORMATION: /product="Beta-ala"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    IleGluGlyProThrLeuArgGlnTrpLeuAlaAlaArgAlaXaaLys    151015    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 9    (D) OTHER INFORMATION: /product="1-Nal"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="Sar"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    IleGluGlyProThrLeuArgGlnXaaLeuAlaAlaArgXaaLys    151015    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 9    (D) OTHER INFORMATION: /product="1-Nal"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 13    (D) OTHER INFORMATION: /product="Ac-Lys"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="Sar"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    IleGluGlyProThrLeuArgGluXaaLeuAlaAlaXaaXaaLys    151015    (2) INFORMATION FOR SEQ ID NO:21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    XaaXaaXaaXaaXaaThrLeuArgGluTrpLeuXaaXaaXaaXaaXaa    151015    XaaXaa    (2) INFORMATION FOR SEQ ID NO:22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    XaaXaaXaaXaaXaaXaaThrLeuArgGluTrpLeuXaaXaaXaaXaa    151015    XaaXaaXaa    (2) INFORMATION FOR SEQ ID NO:23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    XaaXaaXaaXaaXaaThrLeuArgGluTrpLeuXaaXaaXaaXaaXaa    151015    XaaXaa    (2) INFORMATION FOR SEQ ID NO:24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    XaaXaaXaaXaaXaaThrLeuArgGluPheLeuXaaXaaXaaXaaXaa    151015    XaaXaa    (2) INFORMATION FOR SEQ ID NO:25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    ArgGluGlyProThrLeuArgGlnTrpMet    1510    (2) INFORMATION FOR SEQ ID NO:26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    ArgGluGlyProThrLeuArgGlnTrpMet    1510    (2) INFORMATION FOR SEQ ID NO:27:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    SerArgGlyMetThrLeuArgGluTrpLeu    1510    (2) INFORMATION FOR SEQ ID NO:28:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    GluGlyProThrLeuArgGlyTrpLeuAla    1510    (2) INFORMATION FOR SEQ ID NO:29:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    ArgGluGlyGlnThrLeuLysGluTrpLeu    1510    (2) INFORMATION FOR SEQ ID NO:30:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    GluArgGlyProPheTrpAlaLysAlaCys    1510    (2) INFORMATION FOR SEQ ID NO:31:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    ArgGluGlyProArgCysValMetTrpMet    1510    (2) INFORMATION FOR SEQ ID NO:32:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:    CysSerGlyLeuThrLeuArgGluTrpLeuValCys    1510    (2) INFORMATION FOR SEQ ID NO:33:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:    CysLeuThrGlyProPheValThrGlnTrpLeuTyrGluCys    1510    (2) INFORMATION FOR SEQ ID NO:34:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:    CysGlyGluGlyLeuThrLeuThrGlnTrpLeuGluHisCys    1510    (2) INFORMATION FOR SEQ ID NO:35:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:    CysArgAlaGlyProThrLeuLeuGluTrpLeuThrLeuCys    1510    (2) INFORMATION FOR SEQ ID NO:36:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:    CysArgAlaGlyProThrLeuLeuGluTrpLeuThrLeuCys    1510    (2) INFORMATION FOR SEQ ID NO:37:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:    CysArgGlnGlyProThrLeuThrAlaTrpLeuLeuGluCys    1510    (2) INFORMATION FOR SEQ ID NO:38:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:    CysAlaAspGlyProThrLeuArgGluTrpIleSerPheCys    1510    (2) INFORMATION FOR SEQ ID NO:39:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:    CysGluLeuValGlyProSerLeuMetSerTrpLeuThrCys    1510    (2) INFORMATION FOR SEQ ID NO:40:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:    CysGlyThrGluGlyProThrLeuSerThrTrpLeuAspCys    1510    (2) INFORMATION FOR SEQ ID NO:41:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:    CysAspGlnLeuGlyValThrLeuSerArgTrpLeuGluCys    1510    (2) INFORMATION FOR SEQ ID NO:42:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:    SerGlyThrGlyLeuThrLeuArgGluTrpLeuGlySerPheSerLeu    151015    LeuSer    (2) INFORMATION FOR SEQ ID NO:43:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:    CysProGluGlyProThrLeuLeuGlnTrpLeuLysArgGlyTyrSer    151015    SerCys    (2) INFORMATION FOR SEQ ID NO:44:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:    ArgGlyAspGlyProThrLeuSerGlnTrpLeuTyrSerLeuMetIle    151015    MetCys    (2) INFORMATION FOR SEQ ID NO:45:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:    MetValAlaGlyProThrLeuArgGluPheIleAlaSerLeuProIle    151015    HisCys    (2) INFORMATION FOR SEQ ID NO:46:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:    SerMetGlnGlyProThrPheArgGluTrpValSerMetMetLysVal    151015    LeuCys    (2) INFORMATION FOR SEQ ID NO:47:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:    SerValGlnCysGlyProThrLeuArgGlnTrpLeuAlaAlaArgAsn    151015    HisLeuSer    (2) INFORMATION FOR SEQ ID NO:48:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:    GlyAsnAlaAspGlyProThrLeuArgGlnTrpLeuGluGlyArgArg    151015    ProLysAsn    (2) INFORMATION FOR SEQ ID NO:49:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:    SerValArgCysGlyProThrLeuArgGlnTrpLeuAlaAlaArgThr    151015    HisLeuSer    (2) INFORMATION FOR SEQ ID NO:50:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:    LeuAlaIleGluGlyProThrLeuArgGlnTrpLeuHisGlyAsnGly    151015    ArgAspThr    (2) INFORMATION FOR SEQ ID NO:51:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:    HisGlyArgValGlyProThrLeuArgGluTrpLysThrGlnValAla    151015    ThrLysLys    (2) INFORMATION FOR SEQ ID NO:52:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:    CysAlaAspGlyProThrLeuArgGluTrpIleSerPheCys    1510    (2) INFORMATION FOR SEQ ID NO:53:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:    IleSerAspGlyProThrLeuLysGluTrpLeuSerValThrArgGly    151015    AlaSer    (2) INFORMATION FOR SEQ ID NO:54:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:    SerIleGluGlyProThrLeuArgGluTrpLeuThrSerArgThrPro    151015    HisSer    (2) INFORMATION FOR SEQ ID NO:55:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:    ThrIleLysGlyProThrLeuArgGlnTrpLeuLysSerArgGluHis    151015    ThrSer    (2) INFORMATION FOR SEQ ID NO:56:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:    GlyAsnAlaAspGlyProThrLeuArgGlnTrpLeuGluGlyArgArg    151015    ProLysAsn    (2) INFORMATION FOR SEQ ID NO:57:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:    SerIleGluGlyProThrLeuArgGluTrpLeuThrSerArgThrPro    151015    HisSer    (2) INFORMATION FOR SEQ ID NO:58:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:    IleSerAspGlyProThrLeuLysGluTrpLeuSerValThrArgGly    151015    AlaSer    (2) INFORMATION FOR SEQ ID NO:59:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:    CysSerLeuGluAspLeuArgLysArgCys    1510    (2) INFORMATION FOR SEQ ID NO:60:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:    CysArgArgSerGluLeuLeuGluArgCys    1510    (2) INFORMATION FOR SEQ ID NO:61:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:    CysThrPheLysGlnPheLeuAspGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:62:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:    CysThrArgGlyGluTrpLeuArgCysCys    1510    (2) INFORMATION FOR SEQ ID NO:63:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:    CysThrLeuArgGlnTrpLeuGlnGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:64:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:    CysThrLeuGluGluLeuArgAlaCysCys    1510    (2) INFORMATION FOR SEQ ID NO:65:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:    CysThrArgGluGluLeuMetArgLeuCys    1510    (2) INFORMATION FOR SEQ ID NO:66:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:    CysGlnArgAlaAspLeuIleAsnPheCys    1510    (2) INFORMATION FOR SEQ ID NO:67:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:    CysAsnArgAsnAspLeuLeuLeuPheCys    1510    (2) INFORMATION FOR SEQ ID NO:68:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:    CysThrArgThrGluTrpLeuHisGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:69:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 9 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:    CysThrLeuGluPheMetAsnGlyCys    15    (2) INFORMATION FOR SEQ ID NO:70:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:    CysSerLeuGlyGluLeuArgArgLeuCys    1510    (2) INFORMATION FOR SEQ ID NO:71:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:    CysAsnIleAsnGlnLeuArgSerIleCys    1510    (2) INFORMATION FOR SEQ ID NO:72:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:    CysThrMetArgGlnPheLeuValCysCys    1510    (2) INFORMATION FOR SEQ ID NO:73:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:    CysThrArgSerGluTrpLeuGluArgCys    1510    (2) INFORMATION FOR SEQ ID NO:74:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:    CysThrLeuHisGluTyrLeuSerGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:75:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:    CysThrArgGluGluLeuLeuArgGlnCys    1510    (2) INFORMATION FOR SEQ ID NO:76:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:    CysThrPheArgGluPheValAsnGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:77:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:    CysSerArgAlaAspPheLeuAlaAlaCys    1510    (2) INFORMATION FOR SEQ ID NO:78:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:    CysSerCysAlaGlnValValGlnCysCys    1510    (2) INFORMATION FOR SEQ ID NO:79:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:    CysThrLeuArgGlnTrpIleLeuLeuGlyMetCys    1510    (2) INFORMATION FOR SEQ ID NO:80:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:    CysThrLeuArgGluTrpLeuHisGlyGlyPheCys    1510    (2) INFORMATION FOR SEQ ID NO:81:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:    CysThrLeuArgAlaTrpLeuMetSerGluThrCys    1510    (2) INFORMATION FOR SEQ ID NO:82:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:    CysThrLeuArgAlaTrpLeuMetGluSerCysCys    1510    (2) INFORMATION FOR SEQ ID NO:83:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:    CysThrPheGlnValTrpLysLeuAlaArgAsnCys    1510    (2) INFORMATION FOR SEQ ID NO:84:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:    CysLeuLeuArgGluTrpLeuAspXaaArgThrCys    1510    (2) INFORMATION FOR SEQ ID NO:85:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:    CysValLeuArgGluTrpLeuLeuXaaXaaSerCys    1510    (2) INFORMATION FOR SEQ ID NO:86:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:    CysLeuLeuSerGluPheLeuAlaGlyGlnGlnCys    1510    (2) INFORMATION FOR SEQ ID NO:87:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:    CysSerLeuArgGlnTyrLeuAspPheGlyLeuGlySerCys    1510    (2) INFORMATION FOR SEQ ID NO:88:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:    CysThrLeuGlnGluLeuLysGlnSerSerLeuTyrGluCys    1510    (2) INFORMATION FOR SEQ ID NO:89:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:    CysAspLeuSerGluLeuLysThrHisGlyTyrAlaTyrCys    1510    (2) INFORMATION FOR SEQ ID NO:90:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:    CysLysLeuSerAspTrpLeuMetAsnGlyValAlaAlaCys    1510    (2) INFORMATION FOR SEQ ID NO:91:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:    CysSerLeuGlnGluPheLeuSerHisGlyGlyTyrValCys    1510    (2) INFORMATION FOR SEQ ID NO:92:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:    CysSerLeuLysGluPheLeuHisSerGlyLeuMetGlnCys    1510    (2) INFORMATION FOR SEQ ID NO:93:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:    CysThrPheArgGlnLeuLeuGluTyrGlyValSerSerCys    1510    (2) INFORMATION FOR SEQ ID NO:94:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:    CysThrMetArgGluPheLeuValAlaSerGlyValAlaCys    1510    (2) INFORMATION FOR SEQ ID NO:95:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:    CysThrLeuAlaGluPheLeuAlaSerGlyValGluGlnCys    1510    (2) INFORMATION FOR SEQ ID NO:96:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:    CysThrLeuAlaGluPheLeuAlaSerGlyValGluGlnCys    1510    (2) INFORMATION FOR SEQ ID NO:97:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:    CysThrLeuLysGluTrpLeuValSerHisGluValTrpCys    1510    (2) INFORMATION FOR SEQ ID NO:98:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:    CysThrLeuArgGluPheLeuSerLeuGlyMetAsnAlaCys    1510    (2) INFORMATION FOR SEQ ID NO:99:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:    CysThrLeuArgGluPheLeuAspProThrThrAlaValCys    1510    (2) INFORMATION FOR SEQ ID NO:100:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:    CysSerLeuLeuGluPheLeuAlaLeuGlyValAlaLeuCys    1510    (2) INFORMATION FOR SEQ ID NO:101:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:    GlyGlyGlyArgGlyCysThrLeuLysGlnTrpLysGlnGlyAspCys    151015    GlyArgSer    (2) INFORMATION FOR SEQ ID NO:102:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:    CysAsnArgSerGlnLeuLeuAlaAlaCys    1510    (2) INFORMATION FOR SEQ ID NO:103:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:    CysThrLeuGlnGlnTrpLeuSerGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:104:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:    CysThrLeuArgGluPheLysAlaGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:105:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:    CysThrArgAlaGlnPheLeuLysGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:106:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:    CysThrLeuArgGluPheAsnArgGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:107:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:    CysThrLeuSerAspPheLysArgGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:108:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:    CysThrPheArgGlnTrpLysGluAlaCys    1510    (2) INFORMATION FOR SEQ ID NO:109:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:    CysThrLeuSerGluPheArgGlyGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:110:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:    CysThrLeuGlnGluPheLeuGluGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:111:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:    CysThrLeuGlnGlnTrpLysAspGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:112:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:    CysThrArgSerGlnTrpLeuGluGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:113:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:    CysSerLeuGlnGluPheLysHisGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:114:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:    CysThrLeuGlyGluTrpLysArgGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:115:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:    CysThrLeuTrpGlyCysGlyLysArgGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:116:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:    CysThrLeuGlnGluTrpArgGlyGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:117:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:    CysThrArgLeuSerGlyCysTrpLeuCys    1510    (2) INFORMATION FOR SEQ ID NO:118:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118:    CysThrArgThrGlnTrpLeuLeuAspCys    1510    (2) INFORMATION FOR SEQ ID NO:119:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:119:    CysThrLeuAlaGluPheArgArgGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:120:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:120:    CysThrSerThrGlnTrpLeuLeuAlaCys    1510    (2) INFORMATION FOR SEQ ID NO:121:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:121:    CysSerArgSerGlnPheLeuArgSerCys    1510    (2) INFORMATION FOR SEQ ID NO:122:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:122:    CysThrLeuArgGluTrpLeuGluGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:123:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:123:    CysThrLeuArgGluPheLeuLeuMetGlyAlaCys    1510    (2) INFORMATION FOR SEQ ID NO:124:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:124:    CysThrLeuLysGluTrpLeuLeuTrpSerSerCys    1510    (2) INFORMATION FOR SEQ ID NO:125:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:125:    CysThrLeuLeuGluTrpLeuArgAsnProValCys    1510    (2) INFORMATION FOR SEQ ID NO:126:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126:    CysThrLeuArgGlnTrpLeuGlyAspAlaTrpCys    1510    (2) INFORMATION FOR SEQ ID NO:127:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:127:    CysThrLeuGlyGlnTrpLeuGlnMetGlyMetCys    1510    (2) INFORMATION FOR SEQ ID NO:128:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:128:    CysThrLeuArgGluTrpValPheAlaGlyLeuCys    1510    (2) INFORMATION FOR SEQ ID NO:129:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:129:    CysLeuLeuLeuGluPheLeuSerGlyAlaAspCys    1510    (2) INFORMATION FOR SEQ ID NO:130:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:130:    CysThrLeuGlyGluPheLeuAlaGlyHisLeuCys    1510    (2) INFORMATION FOR SEQ ID NO:131:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:131:    CysArgLeuArgGluPheLeuValAspLeuThrCys    1510    (2) INFORMATION FOR SEQ ID NO:132:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:132:    CysSerPheArgSerTrpLeuValAspGlnThrCys    1510    (2) INFORMATION FOR SEQ ID NO:133:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:133:    CysThrLeuArgGluTrpLeuGluAspLeuGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:134:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:134:    CysThrLeuGlnAspTrpLeuValSerTrpThrCys    1510    (2) INFORMATION FOR SEQ ID NO:135:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:135:    CysThrLeuSerGluTrpLeuSerGluLeuSerCys    1510    (2) INFORMATION FOR SEQ ID NO:136:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:136:    CysThrLeuMetGlnTrpLeuGlyGlyTrpProCys    1510    (2) INFORMATION FOR SEQ ID NO:137:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:137:    CysThrLeuArgGluTrpLeuSerTyrGlyThrCys    1510    (2) INFORMATION FOR SEQ ID NO:138:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:138:    CysThrLeuGlnGluTrpLeuSerGlyGlyLeuCys    1510    (2) INFORMATION FOR SEQ ID NO:139:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:139:    GlySerHisGlyCysThrLeuArgGluTrpLeuCysMetLysIleVal    151015    ProCys    (2) INFORMATION FOR SEQ ID NO:140:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:140:    GlnTrpGlnGlyCysThrLeuArgAspCysIleLeuArgGlyValPhe    151015    TrpSer    (2) INFORMATION FOR SEQ ID NO:141:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:141:    SerValAsnSerCysThrLeuArgGluPheLeuThrGlyCysArgVal    151015    PheCys    (2) INFORMATION FOR SEQ ID NO:142:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:142:    SerTyrAspGlyCysThrLeuArgHisTrpLeuMetAspIleTyrGly    151015    AspCys    (2) INFORMATION FOR SEQ ID NO:143:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:143:    GlnArgSerGlyCysThrLeuArgAspTrpValLeuLeuAsnCysLeu    151015    AlaSer    (2) INFORMATION FOR SEQ ID NO:144:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:144:    AsnTyrArgGlyCysThrLeuSerGlnTrpValSerGluGlnIleVal    151015    GlyCys    (2) INFORMATION FOR SEQ ID NO:145:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:145:    GlyArgSerGlyCysThrLeuArgGluTyrLeuGlyGlyMetCysTyr    151015    LeuSer    (2) INFORMATION FOR SEQ ID NO:146:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:146:    AlaSerTrpTyrCysThrValProGluLeuMetGluMetGlnLeuPro    151015    GluCys    (2) INFORMATION FOR SEQ ID NO:147:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:147:    GlySerThrGlyCysThrLeuArgGluXaaLeuHisMetLeuGlyLeu    151015    AspCys    (2) INFORMATION FOR SEQ ID NO:148:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:148:    AlaCysGluGlyCysThrLeuArgGlnTrpLeuGluTyrValArgVal    151015    GlyCys    (2) INFORMATION FOR SEQ ID NO:149:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:149:    AlaGlnArgGlyCysThrLeuGlnTyrPheValSerTyrGlyXaaAsp    151015    MetCys    (2) INFORMATION FOR SEQ ID NO:150:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:150:    GlyValCysGlyCysThrLeuArgGluPheLeuAlaIleProHisThr    151015    SerCys    (2) INFORMATION FOR SEQ ID NO:151:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:151:    SerGluGlyGlyCysThrLeuArgGluTrpValAlaSerSerLeuAla    151015    AsnCys    (2) INFORMATION FOR SEQ ID NO:152:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:152:    SerAsnSerArgCysThrLeuArgGluTrpIleIleGlnGlyCysAsp    151015    PheSer    (2) INFORMATION FOR SEQ ID NO:153:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:153:    SerAsnSerArgCysThrLeuArgGluTrpIleIleGlnGlyCysAsp    151015    PheSer    (2) INFORMATION FOR SEQ ID NO:154:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:154:    CysLeuGlyCysThrLeuSerGlnTrpArgLysArgThrArgCysAsp    151015    ThrHis    (2) INFORMATION FOR SEQ ID NO:155:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:155:    TyrArgGlyCysSerArgAlaGlnLeuLeuGlyGlyGluCysArgLys    151015    Lys    (2) INFORMATION FOR SEQ ID NO:156:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:156:    GlyArgGlyCysThrLeuLysGlnTrpLysGlnGlyAspCysGlyArg    151015    Ser    (2) INFORMATION FOR SEQ ID NO:157:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:157:    ValArgGlyGlyCysAlaLeuArgAspTrpValAlaGlyGluCysPhe    151015    AspTrpThr    (2) INFORMATION FOR SEQ ID NO:158:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:158:    LeuTrpArgGlyCysThrLeuAsnGlyPheLysSerArgHisCysGly    151015    SerProGlu    (2) INFORMATION FOR SEQ ID NO:159:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:159:    CysThrLeuArgSerTrpLysHisArgGlyCysAlaPro    1510    (2) INFORMATION FOR SEQ ID NO:160:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:160:    GlyArgGlyCysThrArgAlaGlnTrpLeuAlaGlyCysCysThrGly    151015    His    (2) INFORMATION FOR SEQ ID NO:161:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:161:    ArgAlaGlyCysThrLeuArgGluPheArgLysGlyCysLeuAlaLeu    151015    (2) INFORMATION FOR SEQ ID NO:162:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:162:    LysArgGlyCysThrLeuAlaGluMetIleArgGlyCysAsnArgSer    151015    Asn    (2) INFORMATION FOR SEQ ID NO:163:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:163:    GlyArgGlyCysThrLeuLysGlnTrpLysGlnGlyAspCysGlyArg    151015    Ser    (2) INFORMATION FOR SEQ ID NO:164:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:164:    ArgTrpArgGlyCysSerLeuAlaLysLeuLysLysGlyAlaAlaCys    151015    GlyArgGly    (2) INFORMATION FOR SEQ ID NO:165:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:165:    ArgGlyGlyCysThrLeuArgGluTrpArgArgValArgValIleAsn    151015    (2) INFORMATION FOR SEQ ID NO:166:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:166:    GlyArgGlyCysThrLeuLysGlnTrpLysGlnGlyAspCysGlyArg    151015    Ser    (2) INFORMATION FOR SEQ ID NO:167:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:167:    ArgTyrGlyCysThrArgHisGlnTrpLeuValGlyThrCysValArg    151015    His    (2) INFORMATION FOR SEQ ID NO:168:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 8 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:168:    GlyProThrLeuArgGlnTrpLeu    15    (2) INFORMATION FOR SEQ ID NO:169:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "oligonucleotide"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:169:    ACCACCTCCGG11    (2) INFORMATION FOR SEQ ID NO:170:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "oligonucleotide"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:170:    TTACTTAGTTA11    (2) INFORMATION FOR SEQ ID NO:171:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "oligonucleotide"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:171:    GAGGTGGTNNKTAACTAAGTAAAGC25    (2) INFORMATION FOR SEQ ID NO:172:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 1    (D) OTHER INFORMATION: /product="Pen"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:172:    XaaAlaAspGlyProThrLeuArgGluTrpIleSerPheCys    1510    (2) INFORMATION FOR SEQ ID NO:173:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:173:    AlaAspGlyProThrLeuArgGluTrpIleSerPheCys    1510    (2) INFORMATION FOR SEQ ID NO:174:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 1    (D) OTHER INFORMATION: /product="Homocys"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:174:    XaaAlaAspGlyProThrLeuArgGluTrpIleSerPheCys    1510    (2) INFORMATION FOR SEQ ID NO:175:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 1    (D) OTHER INFORMATION: /product="D-Cys"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="D-Cys"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:175:    XaaAlaAspGlyProThrLeuArgGluTrpIleSerPheXaa    1510    (2) INFORMATION FOR SEQ ID NO:176:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="D-Cys"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:176:    CysAlaAspGlyProThrLeuArgGluTrpIleSerPheXaa    1510    (2) INFORMATION FOR SEQ ID NO:177:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 1    (D) OTHER INFORMATION: /product="D-Pen"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="D-Cys"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:177:    XaaAlaAspGlyProThrLeuArgGluTrpIleSerPheXaa    1510    (2) INFORMATION FOR SEQ ID NO:178:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 1    (D) OTHER INFORMATION: /product="Homocys"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="Homocys"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:178:    XaaAlaAspGlyProThrLeuArgGluTrpIleSerPheXaa    1510    (2) INFORMATION FOR SEQ ID NO:179:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 13    (D) OTHER INFORMATION: /product="Homocys"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:179:    AlaAspGlyProThrLeuArgGluTrpIleSerPheXaa    1510    (2) INFORMATION FOR SEQ ID NO:180:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 13    (D) OTHER INFORMATION: /product="Pen"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:180:    AlaAspGlyProThrLeuArgGluTrpIleSerPheXaa    1510    (2) INFORMATION FOR SEQ ID NO:181:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:181:    LysAlaAspGlyProThrLeuArgGluTrpIleSerPheGlu    1510    (2) INFORMATION FOR SEQ ID NO:182:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:182:    GluAlaAspGlyProThrLeuArgGluTrpIleSerPheLys    1510    (2) INFORMATION FOR SEQ ID NO:183:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:183:    AlaAspGlyProThrLeuArgGluTrpIleSerPheGlu    1510    (2) INFORMATION FOR SEQ ID NO:184:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 1    (D) OTHER INFORMATION: /product="Pen"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="Pen"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:184:    XaaAlaAspGlyProThrLeuArgGluTrpIleSerPheXaa    1510    (2) INFORMATION FOR SEQ ID NO:185:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:185:    CysIleGluGlyProThrLeuArgGlnTrpLeuAlaAlaArgAla    151015    (2) INFORMATION FOR SEQ ID NO:186:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:186:    CysAlaAspGlyProThrLeuArgGluTrpIleSerPhe    1510    (2) INFORMATION FOR SEQ ID NO:187:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:187:    SerValGlnCysGlyProThrLeuArgGlnTrpLeuAlaAlaArgAsn    151015    HisLeuSer    (2) INFORMATION FOR SEQ ID NO:188:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:188:    MetValGlyProThrLeuArgSerGlyCys    1510    (2) INFORMATION FOR SEQ ID NO:189:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:189:    AspGlyProThrLeuArgGluTrpIleSerPheCys    1510    (2) INFORMATION FOR SEQ ID NO:190:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:190:    GlyProThrLeuArgGluTrpIleSerPheCys    1510    (2) INFORMATION FOR SEQ ID NO:191:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:191:    ProThrLeuArgGluTrpIleSerPheCys    1510    (2) INFORMATION FOR SEQ ID NO:192:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 9 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:192:    ThrLeuArgGluTrpIleSerPheCys    15    (2) INFORMATION FOR SEQ ID NO:193:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:193:    IleGluGlyProThrLeuArgGlnTrpLeuAlaAlaArgAla    1510    (2) INFORMATION FOR SEQ ID NO:194:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:194:    CysIleGluGlyProThrLeuArgGlnTrpLeuAlaAlaArgAlaCys    151015    (2) INFORMATION FOR SEQ ID NO:195:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:195:    IleGluGlyProThrLeuArgGlnTrpLeuAlaAlaArgLys    1510    (2) INFORMATION FOR SEQ ID NO:196:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="Beta-ala"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:196:    IleGluGlyProThrLeuArgGlnTrpLeuAlaAlaArgXaaLys    151015    (2) INFORMATION FOR SEQ ID NO:197:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:197:    IleGluGlyProThrLeuArgGlnTrpLeuAlaAlaLys    1510    (2) INFORMATION FOR SEQ ID NO:198:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 13    (D) OTHER INFORMATION: /product="Beta-ala"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:198:    IleGluGlyProThrLeuArgGlnTrpLeuAlaAlaXaaLys    1510    (2) INFORMATION FOR SEQ ID NO:199:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:199:    GluGlyProThrLeuArgGlnTrpLeuAlaAlaArgAlaLys    1510    (2) INFORMATION FOR SEQ ID NO:200:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="Beta-ala"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:200:    GluGlyProThrLeuArgGlnTrpLeuAlaAlaArgAlaXaaLys    151015    (2) INFORMATION FOR SEQ ID NO:201:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:201:    GluGlyProThrLeuArgGlnTrpLeuAlaAlaArgLys    1510    (2) INFORMATION FOR SEQ ID NO:202:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 13    (D) OTHER INFORMATION: /product="Beta-ala"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:202:    GluGlyProThrLeuArgGlnTrpLeuAlaAlaArgXaaLys    1510    (2) INFORMATION FOR SEQ ID NO:203:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:203:    GluGlyProThrLeuArgGlnTrpLeuAlaAlaLys    1510    (2) INFORMATION FOR SEQ ID NO:204:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 12    (D) OTHER INFORMATION: /product="Beta-ala"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:204:    GluGlyProThrLeuArgGlnTrpLeuAlaAlaXaaLys    1510    (2) INFORMATION FOR SEQ ID NO:205:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 3    (D) OTHER INFORMATION: /product="Sar"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 9    (D) OTHER INFORMATION: /product="1-Nal"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:205:    IleGluXaaProThrLeuArgGlnXaaLeuAlaAlaArgAlaLys    151015    (2) INFORMATION FOR SEQ ID NO:206:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 3    (D) OTHER INFORMATION: /product="Sar"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 9    (D) OTHER INFORMATION: /product="1-Nal"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 15    (D) OTHER INFORMATION: /product="Beta-ala"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:206:    IleGluXaaProThrLeuArgGlnXaaLeuAlaAlaArgAlaXaaLys    151015    (2) INFORMATION FOR SEQ ID NO:207:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 11    (D) OTHER INFORMATION: /product="Ava"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:207:    IleGluGlyProThrLeuArgGlnTrpLeuXaaArgLys    1510    (2) INFORMATION FOR SEQ ID NO:208:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 11    (D) OTHER INFORMATION: /product="Ava"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 13    (D) OTHER INFORMATION: /product="Beta-ala"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:208:    IleGluGlyProThrLeuArgGlnTrpLeuXaaArgXaaLys    1510    (2) INFORMATION FOR SEQ ID NO:209:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="N-methyl-Ala"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:209:    IleGluGlyProThrLeuArgGlnTrpLeuAlaAlaArgXaaLys    151015    (2) INFORMATION FOR SEQ ID NO:210:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="N-methyl-Ala"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 15    (D) OTHER INFORMATION: /product="Beta-ala"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:210:    IleGluGlyProThrLeuArgGlnTrpLeuAlaAlaArgXaaXaaLys    151015    (2) INFORMATION FOR SEQ ID NO:211:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 13    (D) OTHER INFORMATION: /product="p-amino-Phe"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:211:    IleGluGlyProThrLeuArgGlnTrpLeuAlaAlaXaaAlaLys    151015    (2) INFORMATION FOR SEQ ID NO:212:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 13    (D) OTHER INFORMATION: /product="Ac-Lys"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:212:    IleGluGlyProThrLeuArgGlnTrpLeuAlaAlaXaaAlaLys    151015    (2) INFORMATION FOR SEQ ID NO:213:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 7    (D) OTHER INFORMATION: /product="Ac-Lys"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 13    (D) OTHER INFORMATION: /product="Ac-Lys"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:213:    IleGluGlyProThrLeuXaaGlnTrpLeuAlaAlaXaaAlaLys    151015    (2) INFORMATION FOR SEQ ID NO:214:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 9    (D) OTHER INFORMATION: /product="1-Nal"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="Beta-ala"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:214:    IleGluGlyProThrLeuArgGlnXaaLeuAlaAlaArgXaaLys    151015    (2) INFORMATION FOR SEQ ID NO:215:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="Sar"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:215:    IleGluGlyProThrLeuArgGlnTrpLeuAlaAlaArgXaaLys    151015    (2) INFORMATION FOR SEQ ID NO:216:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 9    (D) OTHER INFORMATION: /product="1-Nal"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="Sar"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:216:    IleGluGlyProThrLeuArgGlnXaaLeuAlaAlaArgXaaLys    151015    (2) INFORMATION FOR SEQ ID NO:217:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="Beta-ala"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:217:    IleGluGlyProThrLeuArgGlnPheLeuAlaAlaArgXaaLys    151015    (2) INFORMATION FOR SEQ ID NO:218:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 9    (D) OTHER INFORMATION: /product="1-Nal"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 13    (D) OTHER INFORMATION: /product="Ac-Lys"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="Sar"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:218:    IleGluGlyProThrLeuArgGlnXaaLeuAlaAlaXaaXaaLys    151015    (2) INFORMATION FOR SEQ ID NO:219:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 9    (D) OTHER INFORMATION: /product="1-Nal"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 13    (D) OTHER INFORMATION: /product="Ac-Lys"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="Sar"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:219:    IleGluGlyProThrLeuArgGluXaaLeuAlaAlaXaaXaaLys    151015    (2) INFORMATION FOR SEQ ID NO:220:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 9    (D) OTHER INFORMATION: /product="1-Nal"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 13    (D) OTHER INFORMATION: /product="Ac-Lys"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="Sar"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:220:    IleGluGlyProThrLeuAlaGlnXaaLeuAlaAlaXaaXaaLys    151015    (2) INFORMATION FOR SEQ ID NO:221:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 9    (D) OTHER INFORMATION: /product="1-Nal"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 13    (D) OTHER INFORMATION: /product="Ac-Lys"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="Sar"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:221:    IleGluGlyProThrLeuAlaGluXaaLeuAlaAlaXaaXaaLys    151015    (2) INFORMATION FOR SEQ ID NO:222:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 9    (D) OTHER INFORMATION: /product="Nal"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 13    (D) OTHER INFORMATION: /product="Nle"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="Sar"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:222:    IleGluGlyProThrLeuArgGlnXaaLeuAlaAlaXaaXaaLys    151015    (2) INFORMATION FOR SEQ ID NO:223:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 7    (D) OTHER INFORMATION: /product="Nle"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 9    (D) OTHER INFORMATION: /product="1-Nal"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 13    (D) OTHER INFORMATION: /product="Nle"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="Sar"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:223:    IleGluGlyProThrLeuXaaGlnXaaLeuAlaAlaXaaXaaLys    151015    (2) INFORMATION FOR SEQ ID NO:224:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 11    (D) OTHER INFORMATION: /product="Abu"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 12    (D) OTHER INFORMATION: /product="DipheAla"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:224:    IleGluGlyProThrLeuArgGlnTrpLeuXaaXaaLys    1510    (2) INFORMATION FOR SEQ ID NO:225:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 11    (D) OTHER INFORMATION: /product="Abu"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 12    (D) OTHER INFORMATION: /product="Diphe"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 13    (D) OTHER INFORMATION: /product="Beta-ala"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:225:    IleGluGlyProThrLeuArgGlnTrpLeuXaaXaaXaaLys    1510    (2) INFORMATION FOR SEQ ID NO:226:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 11    (D) OTHER INFORMATION: /product="Abu"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 12    (D) OTHER INFORMATION: /product="DipheAla"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:226:    IleGluGlyProThrLeuArgGlnTrpLeuXaaXaaArgLys    1510    (2) INFORMATION FOR SEQ ID NO:227:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 11    (D) OTHER INFORMATION: /product="Abu"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 12    (D) OTHER INFORMATION: /product="Diphe"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 14    (D) OTHER INFORMATION: /product="Beta-ala"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:227:    IleGluGlyProThrLeuArgGlnTrpLeuXaaXaaArgXaaLeu    151015    (2) INFORMATION FOR SEQ ID NO:228:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 11    (D) OTHER INFORMATION: /product="Abu"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 12    (D) OTHER INFORMATION: /product="DipheAla"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:228:    AlaAspGlyProThrLeuArgGluTrpIleXaaXaaLys    1510    (2) INFORMATION FOR SEQ ID NO:229:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 11    (D) OTHER INFORMATION: /product="Abu"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 12    (D) OTHER INFORMATION: /product="DipheAla"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 13    (D) OTHER INFORMATION: /product="Beta-ala"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:229:    AlaAspGlyProThrLeuArgGluTrpIleXaaXaaXaaLys    1510    (2) INFORMATION FOR SEQ ID NO:230:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 11    (D) OTHER INFORMATION: /product="Abu"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 12    (D) OTHER INFORMATION: /product="DipheAla"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:230:    AlaAspGlyProThrLeuArgGluTrpIleXaaXaaCys    1510    (2) INFORMATION FOR SEQ ID NO:231:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 13    (D) OTHER INFORMATION: /product="Ava"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:231:    AlaAspGlyProThrLeuArgGluTrpIleSerPheXaaAlaAspGly    151015    ProThrLeuArgGluTrpIleSerPhe    2025    (2) INFORMATION FOR SEQ ID NO:232:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:232:    CysIleGluGlyProThrLeuArgGlnTrpLeuAlaAlaArgAlaLys    151015    (2) INFORMATION FOR SEQ ID NO:233:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 93 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..84    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:233:    CTCGAGAGCGGGCAGGTGGTGCATGGGGAGCAGGTGGGTGGTGAGGCC48    LeuGluSerGlyGlnValValHisGlyGluGlnValGlyGlyGluAla    151015    TCCGGGGCCGTTAACGGCCGTGGCCTAGCTGGCCAATAAGTCGAC93    SerGlyAlaValAsnGlyArgGlyLeuAlaGlyGln    2025    (2) INFORMATION FOR SEQ ID NO:234:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:234:    LeuGluSerGlyGlnValValHisGlyGluGlnValGlyGlyGluAla    151015    SerGlyAlaValAsnGlyArgGlyLeuAlaGlyGln    2025    (2) INFORMATION FOR SEQ ID NO:235:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 92 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..63    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:235:    CTCGAGAGCGGGCAGGTGGTGCATGGGGAGCAGGTGGGTGGTGAGGCC48    LeuGluSerGlyGlnValValHisGlyGluGlnValGlyGlyGluAla    151015    TCCGGAGGTGGTNNKTAACTAAGTAAAGCTGGCCAATAAGTCGA92    SerGlyGlyGlyXaa    20    (2) INFORMATION FOR SEQ ID NO:236:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:236:    LeuGluSerGlyGlnValValHisGlyGluGlnValGlyGlyGluAla    151015    SerGlyGlyGlyXaa    20    (2) INFORMATION FOR SEQ ID NO:237:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 122 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..120    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:237:    CAGACTAATTCGAGCTCGAACAACAACAACAATAACAATAACAACAAC48    GlnThrAsnSerSerSerAsnAsnAsnAsnAsnAsnAsnAsnAsnAsn    151015    CTCGGGATCGAGGGAAGGACCGGTCACGTGGCCCGGGAATTCGGATCC96    LeuGlyIleGluGlyArgThrGlyHisValAlaArgGluPheGlySer    202530    TCTAGAGTCGACCTGCAGGCAAGCTT122    SerArgValAspLeuGlnAlaSer    3540    (2) INFORMATION FOR SEQ ID NO:238:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 40 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:238:    GlnThrAsnSerSerSerAsnAsnAsnAsnAsnAsnAsnAsnAsnAsn    151015    LeuGlyIleGluGlyArgThrGlyHisValAlaArgGluPheGlySer    202530    SerArgValAspLeuGlnAlaSer    3540    (2) INFORMATION FOR SEQ ID NO:239:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 51 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..21    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:239:    GGAAGGACCGGAGGTGGTNNKTAACTAAGTAAAGCTGGCCAATAAGTCGAC51    GlyArgThrGlyGlyGlyXaa    15    (2) INFORMATION FOR SEQ ID NO:240:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:240:    GlyArgThrGlyGlyGlyXaa    15    (2) INFORMATION FOR SEQ ID NO:241:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 93 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..84    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:241:    GAAGCGGCGATGGCGGAGCTGAATTACATTCCCCGGTCGCAGGAGGCC48    GluAlaAlaMetAlaGluLeuAsnTyrIleProArgSerGlnGluAla    151015    TCCGGGGCCGTTAACGGCCGTGGCCTAGCTGGCCAATAAGTCGAC93    SerGlyAlaValAsnGlyArgGlyLeuAlaGlyGln    2025    (2) INFORMATION FOR SEQ ID NO:242:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:242:    GluAlaAlaMetAlaGluLeuAsnTyrIleProArgSerGlnGluAla    151015    SerGlyAlaValAsnGlyArgGlyLeuAlaGlyGln    2025    (2) INFORMATION FOR SEQ ID NO:243:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 126 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..96    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:243:    GAAGCGGCGATGGCGGAGCTGAATTACATTCCCCGGTCGCAGGAGGCC48    GluAlaAlaMetAlaGluLeuAsnTyrIleProArgSerGlnGluAla    151015    TCCGGAGGTGGTNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKNNK96    SerGlyGlyGlyXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    202530    TAACTAAGTAAAGCTGGCCAATAAGTCGAC126    (2) INFORMATION FOR SEQ ID NO:244:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 32 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:244:    GluAlaAlaMetAlaGluLeuAsnTyrIleProArgSerGlnGluAla    151015    SerGlyGlyGlyXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    202530    __________________________________________________________________________

What is claimed is:
 1. A compound that binds to a thrombopoietinreceptor, wherein said compound is selected from the group consistingof: ##STR90##
 2. The compound of claim 1, wherein said compound iscovalently attached to a hydrophilic polymer.
 3. The compound of claim2, wherein said hydrophilic polymer has an average molecular weight ofbetween about 500 to about 40,000 daltons.
 4. The compound of claim 2,wherein said hydrophilic polymer has an average molecular weight ofbetween about 5,000 to about 20,000 daltons.
 5. The compound of claim 2,wherein said polymer is selected from the group consisting ofpolyethylene glycol, polypropylene glycol, polylactic acid andpolyglycolic acid.
 6. The compound of claim 5, wherein said compound iscovalently attached to polyethylene glycol.
 7. The compound of claim 1,wherein each of the dimeric subunits of said compound is covalentlyattached to a hydrophilic polymer.
 8. A pharmaceutical compositioncomprising a compound of claim 1 in combination with a pharmaceuticallyacceptable carrier.
 9. A method for treating a patient suffering from adisorder that is susceptible to treatment with a thrombopoietin agonist,comprising administering to the patient a therapeutically effective doseor amount of a compound of claim
 1. 10. A physiologically active,substantially non-immunogenic water soluble polypeptide compositioncomprising a compound of claim 1 coupled with a coupling agent to atleast one polymer having a molecular weight of between about 500 toabout 20,000 daltons selected from the group consisting of polyethyleneglycol and polypropylene glycol, wherein said polymer is unsubstitutedor substituted by alkoxy or alkyl groups, said alkoxy or alkyl groupspossessing less than 5 carbon atoms.
 11. The polypeptide composition inaccordance with claim 10, wherein said polymer has a molecular weight ofabout 750 to about 15,000 daltons.
 12. The polypeptide composition inaccordance with claim 10, wherein said polymer has a molecular weight ofabout 5,000 to about 10,000 daltons.
 13. The polypeptide composition inaccordance with claim 10, wherein said polymer is polyethylene glycol.14. A substantially non-immunogenic water soluble polypeptidecomposition comprising a compound of claim 10 and a pharmaceuticallyacceptable carrier.
 15. A process for preparing a physiologicallyactive, substantially non-immunogenic water soluble polypeptidecomposition from a polypeptide originally having physiological activitycomprising the steps of:(a) reacting at least one terminal carbon atombearing a hydroxy group of a polymer having a molecular weight of fromabout 500 to about 20,000 daltons, wherein said polymer is unsubstitutedor substituted by alkoxy or alkyl groups, said alkoxy or alkyl groupspossessing less than 5 carbon atom; and (b) reacting a physiologicallyactive polypeptide with from 10 to 100 moles of said activated polymerper mole of polypeptide by coupling said to the reactive terminal groupof said polymer to provide said physiologically active, substantiallynon-immunogenic water soluble polypeptide composition.
 16. The processof claim 15, wherein said polymer is polyethylene glycol.
 17. Theprocess of claim 15, wherein said coupling agent is cyanuric chloride.18. A compound that binds to a thrombopoietin receptor, wherein saidcompound is selected from the group consisting of: ##STR91##
 19. Acompound that binds to a thrombopoietin receptor, wherein said compoundis selected from the group consisting of: ##STR92## wherein n is aninterger having a value ranging from about 10 to about
 1000. 20. Aphysiologically active, substantially non-immunogenic water solublepolypeptide composition comprising a polypeptide having a molecularweight of 15,000 daltons or less coupled with a coupling agent to atleast one polymer having a molecular weight of between about 500 toabout 20,000 daltons selected from the group consisting of polyethyleneglycol and polypropylene glycol, wherein said polymer is unsubstitutedor substituted by alkoxy or alkyl groups, said alkoxy or alkyl groupspossessing less than 5 carbon atoms.